Methods and Compositions for Treating Idiopathic Pulmonary Fibrosis

ABSTRACT

Provided is a pharmaceutical composition comprising an effective amount of itraconazole, and a pharmaceutically acceptable excipient. The use of the pharmaceutical composition for treatment of idiopathic pulmonary fibrosis is also provided.

BACKGROUND OF THE INVENTION

Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive lungdisease that results in respiratory failure and death. IPF is the mostcommon cause of death from progressive lung disease, and affects about 5million people worldwide. An estimated median survival after diagnosisis only 2-3 years (Chakraborty et al., (2014) Expert Opin InvestigDrugs, 23:893-910; Spagnolo et al., (2015) Pharmacology & Therapeutics152:18-27; Tzouvelekis et al., (2015) Therapeutics and Clinical RiskManagement 11:359-370). In the United States, as many as 89,000 peopleare afflicted with IPF, with about 34,000 newly diagnosed annually(Raghu G et al., (2006) Am J Respir Crit Care Med 174: (7):810-816).Prevalence of IPF ranges from 14.0 to 42.7 cases per 100,000 persons andthe annual incidence ranges from 6.8 to 16.3 cases per 100,000 persons,depending on the strictness of the diagnostic criteria employed (Raghu Get al., supra.). The prevalence of IPF increases with age, with most IPFpatients at the age of 60 years or even older at the time of diagnosis.The disease is more common in men than in women (Fernandez Perez E R etal., (2010) Chest 137(1):129-137), with most patients being current orformer smokers. A familial form of IPF may account for as many as 20% ofIPF cases (Loyd J E, (2008) Eur Respir Rev 17(109):163-167).

The etiology of IPF remains unknown. Potential factors, such ascigarette smoking, dust exposure and infection agents, however, havebeen associated with the development of IPF. IPF is characterized byprogressive and irreversible distortion of the lung's architecture as aresult of apoptosis of epithelial and endothelial cells, fibroblasthyperplasia and extracellular metric remodeling (Chakraborty et al.,(2014) Expert Opin Investig Drugs, 23:893-910). As interstitial fibrosisadvances with accompanying distortion of lung architecture, the lungbecomes less compliant, increasing the effort associated with breathing,leading to dyspnea. Typically, lung function declines slowly over time,but some patients experience rapid declines that can lead tohospitalization or death, particularly in later stages of the disease.

Development of agents for treatment of IPF has been slow in progress.The first two agents for treating IPF, pirfenidone and nintedanib, wereapproved only at the end of 2014 (King et al., (2014) N Engl J Med370:2083-92; Richeldi et al., (2014) N Engl J Med 370:2071-82). Thesetwo agents, however, have only limited efficacy and significant sideeffects, and require complicated dosing regimen. Recently conductedphase 3 clinical trials of pirfenidone, sildenafil, bosentan,etanercept, and interferon gamma-1b failed to demonstrate efficacy intheir primary endpoints. N-acetyl cysteine (NAC), corticosteroids, andthe immunosuppressive drugs cyclophosphamide and azathioprine arecommonly prescribed, but there is little evidence that use of thesedrugs improves patient outcome or alters the natural course of thedisease (Collard H R et al., (2004) Chest 125(6):2169-2174; Walter N etal., (2006) Proc Am Thorac Soc 3(4):377-381). In fact, the combinationof prednisone, azathioprine, and NAC produced a worse outcome than NACor placebo in a recent IPF study (NIH News, Oct. 24, 2011). Lungtransplantation is the only treatment that improves survival (Walter Net al, supra.), but most IPF patients are not eligible fortransplantation because of their age or comorbid conditions. IPFpatients usually are managed with supportive measures such assymptomatic treatment of cough and dyspnea, supplemental oxygen forhypoxemia, smoking cessation, pulmonary rehabilitation, and prophylaxisand control of respiratory tract infections.

The progressive and fatal course of IPF coupled with the absence ofapproved drugs underscore the need for new methods and agents to treatthis devastating disease. The present invention meets this unmet medicalneed by providing novel methods and agents for use in treating IPF.

Itraconazole is an imidazole/triazole type antifungal agent. Recently,Bollong et al. (2017) described an image-based assay of screening fornovel anti-fibrotic compounds, and identified itraconazole to be active(Bollong et al., (2017) PNAS, 114 (18): 4679-4684). However, in U.S.Patent Publication No. 20170362211, the same group of researcherspointed out that, even though itraconazole may have efficacy in bothbleomycin-induced lung and carbon tetrachloride-induced liver fibrosismouse models, the drug's use as an anti-fibrotic is limited due to knownadverse effects, such as P450 inhibition.

Itraconazole is known to be a highly selective inhibitor of cytochromeP-450 sterol C-14 α-demethylation (Perfect J R, (2017) Nature ReviewDrug Discovery 16:603-616), and to have inhibitory activity toward boththe hedgehog signaling pathway (Kim J et al., (2010) Cancer Cell.17:388-399; Horn A et al., (2012) Arthritis Rheum. 64:2724-2733; BolanosA L et al., (2012) Am J Physiol Lung Cell Mol Physiol. 303:L978-L990)and angiogenesis (Chong et al., (2007) ACS Chem Biol. 2:263-70).Itraconazole was also reported to have inhibitory activities in thevascular endothelial growth factor receptor 2 signaling in endothelialcells (Nacev B A et al., (2011) J Biol Chem. 286:44045-44056; ChaudharyN I et al., (2007) EurRespir J. 29:976-985). As itraconazole is an FDAapproved drug with a well characterized safety and tolerance profile,researchers named as inventors of US20170362211 apparently have foundthat the doses and/or blood levels of itraconazole needed for fibrosistreatment exceeded the safety profile/doses approved by FDA. Theseresearchers instead directed their further efforts to developing a newclass of derivatives of itraconazole for the treatment of multiplefibrosis related diseases.

SUMMARY OF THE INVENTION

The present inventors have surprisingly discovered that with a suitabledosing regimen or a novel administration route, itraconazole can be usedto prevent or treat IPF in an effective and safe manner.

Accordingly, in one embodiment, the present invention provides a methodfor treating idiopathic pulmonary fibrosis, by administering to apatient in need thereof, a pharmaceutical composition comprising aneffective amount of itraconazole, and a pharmaceutically acceptableexcipient. In one embodiment, the daily dose of itraconazole is in therange of 20 mg to 1200 mg for an adult human patient.

In one embodiment, the method of the invention comprises administeringitraconazole in combination with an effective amount of one or moreknown antifibrosis agents, e.g. pirfenidone and nintedanib.

In one embodiment, the method of the invention comprises administering adaily dose of itraconazole of 0.5 mg/kg to 200 mg/kg bodyweight.Itraconazole may be administered by any suitable means for oral,parenteral, rectal, cutaneous, nasal, vaginal, or inhalant use.

In one embodiment, the pharmaceutical composition is delivered using aninhaler directly into the lungs of the patient, for example, at a levelthat is less than about 1/10 of an oral dosage. In another embodiment,the pharmaceutical composition is in a dosage form of a spray, or anebulizer.

Also provided are pharmaceutical compositions for treating IPFcomprising an amount of itraconazole effective for treating IPF, and apharmaceutically acceptable excipient. In one embodiment, the daily doseof itraconazole is in the range of 20 mg to 1200 mg.

Although inhalable formulations of itraconazole is known, see e.g. U.S.Pat. No. 9,061,027, and review by Le and Schiller (Le and Schiller,(2010) Curr Fungal Infect Rep. 4:96-102), they were only formulated foranti-fungal purposes, and had not been formulated for long-term, lowdose usages as required for IPF treatment or prophylactics. Using a newnanotechnology technique that spray-freezes a drug with poor watersolubility into a liquid, the effectiveness of aerosolized itraconazoleas a prophylactic agent against invasive pulmonary aspergillosis causedby Aspergillus flavus and Aspergillus fumigatus was studied inimmunocompromised mice (Alvarez et al., (2007) J Infect 55:68-74; Hoebenet al., (2006) Antimicrob Agents Chemother. 50:1552-1554). Single andmultiple aerosolized dose studies in mice have demonstrated the abilityto achieve effective anti-fungal pulmonary concentrations within 60 minafter completion of nebulization while maintaining serum levels 25 to 50times lower (McConville et al., (2006) Pharm Res. 23:901-911; Vaughn etal., (2006) Eur J Pharm Biopharm. 63:95-102).

Nevertheless, although these antifungal results appear promising inmice, the authors consistently cautioned that further studies are neededbefore extrapolating them to the clinical setting. Anti-fibrotictreatment generally lasts for months, or years, or even for life, whileanti-fungal treatments last for at most a few weeks. Therefore, theconcerns for any side effect of an anti-fibrotic drug is greatlyexacerbated, and must be confronted.

Accordingly, in one embodiment, the present invention provides a dosageform for delivering itraconazole for treating IPF in a patient in needthereof, wherein the dosage form directly delivers an effective amountof itraconazole into the lungs of the patient. The dosage form can be aspray or a nebulizer, and delivers less than about 1/10 of an oraldosage for to the patient.

In one embodiment, the dosage form further comprises a pharmaceuticallyeffective amount of an antifibrosis agent, which can be pirfenidone ornintedanib.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows mice body weight changes following bleomycin plusnintedanib or fluconazole treatment.

FIG. 2 shows BALF collagen levels following bleomycin plus nintedanib orfluconazole treatment.

FIG. 3 shows total cell number (left four columns) and macrophage number(right four columns) in BALFs following bleomycin plus nintedanib orfluconazole treatment.

FIG. 4 shows inflammation area (panel A), modified Ashcroft score (panelB) and α-SMA staining (panel C) of mice lungs following bleomycin plusnintedanib or fluconazole treatment.

FIG. 5 shows mice body weight changes following bleomycin treatment plusitraconazole, nintedanib or combination administration.

FIG. 6 shows BALF collagen levels following bleomycin treatment plusitraconazole, nintedanib or combination administration.

FIG. 7 shows cell counts in BALFs following bleomycin treatment plusitraconazole, nintedanib or combination administration.

FIG. 8 shows inflammation area (panel A), modified Ashcroft score (panelB) and α-SMA staining (panel C) of mice lungs following bleomycintreatment plus itraconazole, nintedanib or combination administration.

FIG. 9 shows total cell number (panel A) and neutrophil number (panel B)in BALFs following bleomycin treatment plus inhalation or oraladministration of itraconazole, or nintedanib.

FIG. 10 shows TGF-β1 levels (panel A) and collagen type I levels (panelB) in BALFs following bleomycin treatment plus inhalation or oraladministration of itraconazole, or nintedanib.

FIG. 11 shows hydroxyproline levels in lungs following bleomycintreatment plus inhalation or oral administration of itraconazole, ornintedanib.

FIG. 12 shows Ashcroft scores of mice lung tissues following bleomycintreatment plus inhalation or oral administration of itraconazole, ornintedanib.

FIG. 13 shows Masson IODs in the lung tissues following bleomycintreatment plus inhalation or oral administration of itraconazole, ornintedanib.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the following description, specific details are set forth inorder to provide a more thorough understanding of the invention.However, the invention may be practiced without these particulars. Inother instances, well known elements have not been shown or described indetail to avoid unnecessarily obscuring the invention. Accordingly, thespecification and drawings are to be regarded in an illustrative, ratherthan a restrictive, sense.

The present invention relates to methods and compositions for treatingIPF in humans. The terms “treat” and “treatment” are used broadly todenote therapeutic and prophylactic interventions that favorably alter apathological state. Treatments include procedures that moderate orreverse the progression of, reduce the severity of, prevent, or cure adisease. As used herein, the term “IPF” or “idiopathic pulmonaryfibrosis” includes all forms of idiopathic pulmonary fibrosis, such asoccupational and environmental, auto-immune, scleroderma, sarcoidosis,drug- and radiation-induced, and genetic/familial fibrosis.

The amount of itraconazole administered can vary with the patient, theroute of administration and the result sought. Optimum dosing regimensfor particular patients can be readily determined by one skilled in theart. For example, the daily dose of itraconazole can be from about 20 mgto about 1200 mg. In one embodiment, the daily dose of itraconazole maybe in the range of 0.5 milligrams per kilogram of body weight to 200milligrams per kilogram of body weight.

According to a certain embodiment of the present invention, there isprovided an itraconazole formulation composition for oral pulmonary orintranasal inhalation delivery, comprising formulations for aerosoladministration of itraconazole for the prevention or treatment ofidiopathic pulmonary fibrosis. According to a certain embodiment of thepresent invention, there is provided methods of administeringitraconazole to a patient in need thereof, or a method for treating IPFof a patient in need thereof, comprising using the inhalationformulation of the present invention.

Itraconazole can be administered in the form of a pharmaceuticalcomposition together with a pharmaceutical carrier. The pharmaceuticalcomposition can be in dosage unit form such as powder, syrup,suspension, emulsion, solution, gel including hydrogel, spray oraerosol, or the like. Sustained release formulations can also be used.

A large variety of delivery vehicles for administering the compositionare contemplated as within the scope of the present invention whencontaining therapeutic amounts of itraconazole. Suitable deliveryvehicles include, but are not limited to, microcapsules or microspheres;liposomes and other lipid-based release systems; absorbable and/orbiodegradable mechanical barriers, polymeric or gel-like materials.

In some embodiments, the dosage form may provide a dosage of between 20to 1200 mg of itraconazole. The dosage form may also be formulated toprovide a daily dosage in the range of 1-20 mg per kilogram of bodyweight.

The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice. Sustained release formulations canalso be used. Itraconazole may be formulated in a variety of ways thatare known in the art, e.g. as liquid.

In accordance with the invention, the pharmaceutical composition of thepresent invention is an effective treatment for IPF and provides aneffective means of delaying disease progression associated withfibrosis. The composition in inhaler, for example, can be more effectivethan an oral dosage, with fewer side effects. Lower doses can be used,reducing the overall side effect burden.

Inhalable Formulation/Aerosol Delivery

Itraconazole is preferably directly administered as an aerosol to a siteof IPF pathology.

Several device technologies exist to deliver either dry powder or liquidaerosolized products. Dry powder formulations generally require lesstime for drug administration, yet longer and more expensive developmentefforts. Conversely, liquid formulations have historically suffered fromlonger administration times, yet have the advantage of shorter and lessexpensive development efforts.

The solubility of itraconazole in 0.1N HCl is approximately 4-6 μg/mLand in water is 1-4 ng/mL. Itraconazole exhibits very poor oralbioavailability owing to its insolubility in intestinal fluids.

Accordingly, in one embodiment, a particular formulation of itraconazoledisclosed herein is combined with a particular aerosolizing device toprovide an aerosol for inhalation that is optimized for maximum drugdeposition at a desired site. Factors that can be optimized includesolution or solid particle formulation, rate of delivery, and particlesize and distribution produced by the aerosolizing device.

Particle Size and Distribution

The distribution of aerosol particle/droplet size can be expressed interms of either: the mass median aerodynamic diameter (MMAD)—the dropletsize at which half of the mass of the aerosol is contained in smallerdroplets and half in larger droplets; volumetric mean diameter (VMD);mass median diameter (MMD); or the fine particle fraction (FPF)—thepercentage of particles that are <5 μm in diameter. These measurementsmay be made by impaction (MMD and MMAD) or by laser (VMD). For liquidparticles, VMD, MMD and MMAD may be the same if environmental conditionsare maintained, e.g., standard humidity. However, if humidity is notmaintained, MMD and MMAD determinations will be smaller than VMD due todehydration during impactor measurements. For the purposes of thisdescription, VMD, MMD and MMAD measurements are considered to be understandard conditions such that descriptions of VMD, MMD and MMAD will becomparable. Similarly, dry powder particle size determinations in MMDand MMAD are also considered comparable.

These measures have been used for comparisons of the in vitroperformance of different inhaler device and drug combinations. Ingeneral, the higher the fine particle fraction, the higher theproportion of the emitted dose that is likely to deposit in the lung.

Generally, inhaled particles are subject to deposition by one of twomechanisms: impaction, which usually predominates for larger particles,and sedimentation, which is prevalent for smaller particles. Impactionoccurs when the momentum of an inhaled particle is large enough that theparticle does not follow the air stream and encounters a physiologicalsurface. In contrast, sedimentation occurs primarily in the deep lungwhen very small particles which have traveled with the inhaled airstream encounter physiological surfaces as a result of random diffusionwithin the air stream.

For pulmonary administration, the upper airways are avoided in favor ofthe middle and lower airways. Pulmonary drug delivery may beaccomplished by inhalation of an aerosol through the mouth and throat.Particles having a mass median aerodynamic diameter (MMAD) of greaterthan about 5 microns generally do not reach the lung; instead, they tendto impact the back of the throat and are swallowed and possibly orallyabsorbed. Particles having diameters of about 1 to about 5 microns aresmall enough to reach the upper- to mid-pulmonary region (conductingairways), but are too large to reach the alveoli. Smaller particles,i.e., about 0.5 to about 2 microns, are capable of reaching the alveolarregion. Particles having diameters smaller than about 0.5 microns canalso be deposited in the alveolar region by sedimentation, although verysmall particles may be exhaled.

In some embodiments, the particle size of the aerosol is optimized tomaximize itraconazole deposition at the site of pulmonary pathology, andto maximize tolerability.

Intolerability (e.g., cough and bronchospasm) may occur from upperairway deposition from both inhalation impaction of large particles andsettling of small particles during repeated inhalation and expiration.Thus, in one embodiment, an optimum particle size is used (e.g.,MMAD=2-5 μm) in order to maximize deposition at a mid-lung and tominimize intolerability associated with upper airway deposition.Moreover, generation of a defined particle size with limited geometricstandard deviation (GSD) may optimize deposition and tolerability.Narrow GSD limits the number of particles outside the desired MMAD sizerange.

In one embodiment, an aerosol containing itraconazole disclosed hereinis provided having a MMAD from about 2 microns to about 5 microns with aGSD of less than or equal to about 2.5 microns. In another embodiment,an aerosol having an MMAD from about 2.8 microns to about 4.3 micronswith a GSD less than or equal to 2 microns is provided. In anotherembodiment, an aerosol having an MMAD from about 2.5 microns to about4.5 microns with a GSD less than or equal to 1.8 microns is provided.

In some embodiments, itraconazole that is intended for respiratorydelivery can be administered as aqueous formulations, as suspensions orsolutions in halogenated hydrocarbon propellants, or as dry powders.Aqueous formulations may be aerosolized by liquid nebulizers employingeither hydraulic or ultrasonic atomization. Propellant-based systems mayuse suitable pressurized metered-dose inhalers (pMDIs). Dry powders mayuse dry powder inhaler devices (DPIs), which are capable of dispersingthe drug substance effectively. A desired particle size and distributionmay be obtained by choosing an appropriate device.

Lung Deposition as used herein, refers to the fraction of the nominaldose of an active pharmaceutical ingredient (API) that is bioavailableat a specific site of pharmacologic activity upon administration of theagent to a patient via a specific delivery route. For example, a lungdeposition of 30% means 30% of the active ingredient in the inhalationdevice just prior to administration is deposited in the lung. Likewise,a lung deposition of 60% means 60% of the active ingredient in theinhalation device just prior to administration is deposited in the lung,and so forth. Lung deposition can be determined using methods ofscintigraphy or deconvolution. In some embodiments, the presentinvention provides for methods and inhalation systems for the treatmentor prophylaxis of a respiratory condition in a patient, comprisingadministering to the patient a nominal dose of itraconazole with aliquid nebulizer. In some embodiments, the liquid nebulizer is a highefficiency liquid nebulizer. In some embodiments, a lung deposition ofitraconazole of at least about 7%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, or at leastabout 85%, based on the nominal dose of itraconazole is achieved.

There are two main methods used to measure aerosol deposition in thelungs. First, γ-scintigraphy is performed by radiolabeling the drug witha substance like 99m-technetium, and scanning the subject afterinhalation of the drug. This technique has the advantage of being ableto quantify the proportion of aerosol inhaled by the patient, as well asregional distribution in the upper airway and lungs. Second, since mostof the drug deposited in the lower airways will be absorbed into thebloodstream, pharmacokinetic techniques are used to measure lungdeposition. This technique can assess the total amount of ICSs thatinteracts with the airway epithelium and is absorbed systemically, butwill miss the small portion that may be expectorated or swallowed aftermucociliary clearance, and cannot tell us about regional distribution.Therefore, γ-scintigraphy and pharmacokinetic studies are in many casesconsidered complementary.

In some embodiments, administration of itraconazole with a liquidnebulizer provides a GSD of emitted droplet size distribution of about1.0 μm to about 2.5 μm, about 1.2 μm to about 2.0 μm, or about 1.0 μm toabout 2.0 μm. In some embodiments, the MMAD is about 0.5 μm to about 5μm, or about 1 to about 4 μm or less than about 5 μm. In someembodiments, the VMD is about 0.5 μm to about 5 μm, or about 1 to about4 μm or less than about 5 μm.

The Delivered Dose (DD) of drug to a patient is the certain portion ofvolume of liquid filled into the nebulizer, i.e. the fill volume, whichis emitted from the mouthpiece of the device. The difference between thenominal dose and the DD is the amount of volume lost primarily toresidues, i.e. the amount of fill volume remaining in the nebulizerafter administration, or is lost in aerosol form during expiration ofair from the patient and therefore not deposited in the patient's body.In some embodiments, the DD of the nebulized formulations describedherein is at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, or at least about 80%.

The Respirable Delivered Dose (RDD) is an expression of the deliveredmass of drug contained within emitted droplets from a nebulizer that aresmall enough to reach and deposit on the surface epithelium of thepatients lung. The RDD is determined by multiplying the DD by the FPF.

In some embodiments, administration of an aqueous inhalationitraconazole solution with a liquid nebulizer provides an RDD of atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, or at leastabout 80%.

In one embodiment, described herein is an aqueous droplet containingitraconaxole, wherein the aqueous droplet has a diameter less than about5.0 μm. In some embodiments, the aqueous droplet has a diameter lessthan about 5.0 μm, less than about 4.5 μm, less than about 4.0 μm, lessthan about 3.5 μm, less than about 3.0 μm, less than about 2.5 μm, lessthan about 2.0 μm, less than about 1.5 μm, or less than about 1.0 μm.

In some embodiments, the aqueous droplet further comprises one or moreco-solvents. In some embodiments, the one or more co-solvents areselected from ethanol and propylene glycol. In some embodiments, theaqueous droplet further comprises a buffer. In some embodiments, thebuffer is a citrate buffer or a phosphate buffer. In some embodiments,the droplet was produced from a liquid nebulizer and an aqueous solutionof itraconazole as described herein. In some embodiments, the aqueousdroplet is produced from an aqueous solution that has concentration ofitraconazole between about 0.1 mg/mL and about 60 mg/mL.

Also described are aqueous aerosols comprising a plurality of aqueousdroplets of itraconazole as described herein.

In some embodiments, at least about 30% of the aqueous droplets in theaerosol have a diameter less than about 5 μm. In some embodiments, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, or at least about 90% of the aqueous droplets in the aerosol have adiameter less than about 5 μm. In some embodiments, the aqueous aerosolsare produced with a liquid nebulizer. In some embodiments, the aqueousaerosols are produced with a high efficiency liquid nebulizer.

Liquid Nebulizer

In one embodiment, a nebulizer is selected on the basis of allowing theformation of an aerosol of an itraconazole disclosed herein having anMMAD predominantly between about 1 to about 5 microns. In oneembodiment, the delivered amount of itraconazole provides a therapeuticeffect for IPF pathology.

Two types of nebulizers, jet and ultrasonic, are able to produce anddeliver aerosol particles having sizes between 2 and 4 micron. Theseparticle sizes have been shown as being optimal for middle airwaydeposition. However, unless a specially formulated solution is used,these nebulizers typically need larger volumes to administer sufficientamount of drug to obtain a therapeutic effect. A jet nebulizer utilizesair pressure breakage of an aqueous solution into aerosol droplets. Anultrasonic nebulizer utilizes shearing of the aqueous solution by apiezoelectric crystal. Typically, however, the jet nebulizers are onlyabout 10% efficient under clinical conditions, while the ultrasonicnebulizer is only about 5% efficient. The amount of pharmaceuticaldeposited and absorbed in the lungs is thus a fraction of the 10% inspite of the large amounts of the drug placed in the nebulizer. Theamount of drug that is placed in the nebulizer prior to administrationto the mammal is generally referred to the “nominal dose,” or “loadeddose.” The volume of solution containing the nominal dose is referred toas the “fill volume.” Smaller particle sizes or slow inhalation ratespermit deep lung deposition. Both middle-lung and alveolar depositionmay be desired for this invention depending on the indication, e.g.,middle and/or alveolar deposition for pulmonary fibrosis and systemicdelivery. Exemplary disclosure of compositions and methods forformulation delivery using nebulizers can be found in, e.g., US2006/0276483, including descriptions of techniques, protocols andcharacterization of aerosolized mist delivery using a vibrating meshnebulizer.

Accordingly, in one embodiment, a vibrating mesh nebulizer is used todeliver in preferred embodiments an aerosol of itraconazole as disclosedherein. A vibrating mesh nebulizer comprises a liquid storage containerin fluid contact with a diaphragm and inhalation and exhalation valves.In one embodiment, about 1 to about 6 mL of itraconazole formulation isplaced in the storage container and the aerosol generator is engagedproducing atomized aerosol of particle sizes selectively between about 1and about 5 micron. In one embodiment, about 1 to about 10 mL ofitraconazole formulation is placed in the storage container and theaerosol generator is engaged producing atomized aerosol of particlesizes selectively between about 1 and about 5 micron. In one embodiment,about the volume of itraconazole formulation that is originally placedin the storage container and the aerosol generator is replaced toincrease the administered dose size.

In some embodiments, an itraconazole formulation as disclosed herein, isplaced in a liquid nebulization inhaler and prepared in dosages todeliver from about 34 mg to about 463 mg from a dosing solution of about0.5 to about 6 mL with MMAD particles sizes between about 1 to about 5micron being produced.

By non-limiting example, a nebulized itraconazole may be administered inthe described respirable delivered dose in less than about 20 min, lessthan about 15 min, less than about 10 min, less than about 7 min, lessthan about 5 min, less than about 3 min, or less than about 2 min.

By non-limiting example, a nebulized itraconazole may be administered inthe described respirable delivered dose using a breath-actuatednebulizer in less than about 20 min, less than about 10 min, less thanabout 7 min, less than about 5 min, less than about 3 min, or less thanabout 2 min.

By non-limiting example, in other circumstances, a nebulizeditraconazole may achieve improved tolerability and/or exhibit anarea-under-the-curve (AUC) shape-enhancing characteristic whenadministered over longer periods of time. Under these conditions, thedescribed respirable delivered dose in more than about 2 min, preferablymore than about 3 min, more preferably more than about 5 min, morepreferably more than about 7 min, more preferably more than about 10min, and in some cases most preferable from about 10 to about 20 min.

In one embodiment, itraconazole is formulated to permit mist, gas-liquidsuspension or liquid nebulized, dry powder and/or metered-dose inhaledaerosol administration to supply effective concentrations or amountsconferring desired anti-fibrotic or tissue-remodeling benefits, forinstance, to prevent, manage or treat patients with pulmonary fibrosis.

Any known inhalation nebulizer suitable to provide delivery of amedicament as described herein may be used in the various embodimentsand methods described herein. Such nebulizers include, e.g., jetnebulizers, ultrasonic nebulizers, pulsating membrane nebulizers,nebulizers with a vibrating mesh or plate with multiple apertures, andnebulizers comprising a vibration generator and an aqueous chamber.Examples of commercially available nebulizers suitable for use in thepresent invention are described, inter alia, in U.S. Pat. No.10,105,356, which is incorporated herein by reference in its entirety.

In one aspect, described herein is a method for the treatment ofidiopathic pulmonary fibrosis in a mammal comprising administering adose of itraconazole by inhalation to the mammal in need thereof on achronic dosing schedule. In some embodiments, the continuous dosingschedule includes administering a dose of itraconazole daily, everyother day, every third day, every fourth day, every fifth day, everysixth day, weekly, biweekly, monthly or bimonthly. In some embodiments,the dosing schedule, whether daily or less than daily, includesadministering one, two, three, or more than three doses of itraconazoleon the days of dosing.

In some embodiments, each inhaled dose of itraconazole is administeredwith a nebulizer, a metered dose inhaler, or a dry powder inhaler. Insome embodiments, each inhaled dose comprises a solution, e.g. anaqueous solution, and/or an ethanol solution of itraconazole. In someembodiments, each inhaled dose comprises from about 0.4 mL to about 240mL of an aqueous solution of itraconazole, wherein the concentration ofitraconazole in the aqueous solution is from about 0.1 mg/mL to about 60mg/mL, such as 5 mg/mL to 50 mg/mL.

In some embodiments, the solution of each inhaled dose further comprisesone or more additional ingredients selected from co-solvents, tonicityagents, sweeteners, surfactants, wetting agents, chelating agents,anti-oxidants, salts, and buffers. In some embodiments, the aqueoussolution of each inhaled dose may further comprise ethanol, a citratebuffer or phosphate buffer, and one or more salts selected from thegroup consisting of sodium chloride, magnesium chloride, sodium bromide,magnesium bromide, calcium chloride and calcium bromide.

In some embodiments, the aqueous solution of each inhaled dosecomprises: water, ethanol, sodium carboxymethyl cellulose, orDMSO/PEG400; itraconazole at a concentration from about 5 mg/mL to about50 mg/mL; one or more salts, wherein the total amount of the one or moresalts is from about 0.01% to about 2.0% by weight of the weight ofaqueous solution; and optionally a phosphate buffer that maintains thepH of the solution from about pH 5.0 to about pH 8.0, or citrate bufferthan maintains the pH of the solution from about 4.0 to about 7.0.

In some embodiments, each inhaled dose is administered with a liquidnebulizer. In some embodiments, the inhaled doses are delivered bynebulization using standard tidal breathing of continuous flow aerosolor breath actuated aerosol.

In some embodiments, the liquid nebulizer: (i) after administration ofthe inhaled dose, achieves lung deposition of at least 7% of theitraconazole administered to the mammal; (ii) provides a GeometricStandard Deviation (GSD) of emitted droplet size distribution of theaqueous solution of about 1.0 μm to about 2.5 μm; (iii) provides: a) amass median aerodynamic diameter (MMAD) of droplet size of the aqueoussolution emitted with the high efficiency liquid nebulizer of about 1 μmto about 5 μm; b) a volumetric mean diameter (VMD) of about 1 μm toabout 5 μm; and/or c) a mass median diameter (MMD) of about 1 μm toabout 5 μm; or (iv) provides a fine particle fraction (FPF=% ≤5 μm) ofdroplets emitted from the liquid nebulizer of at least about 30%. Insome embodiments, the liquid nebulizer provides an output rate of atleast 0.1 mL/min; or provides at least about 25% of the aqueous solutionto the mammal.

In some embodiments, a) the lung tissue Cmax of itraconazole from eachinhaled dose is at least equivalent to or greater than a lung tissueCmax of up to 801 mg of an orally administered dosage of itraconazole;and/or b) the blood AUC₀₋₂₄ of itraconazole from each inhaled dose thatis directly administered to the lungs of the mammal is less than orequivalent to the blood AUC₀₋₂₄ of up to 801 mg of an orallyadministered dosage of itraconazole. In some embodiments, the bloodAUC₀₋₂₄ of itraconazole from each inhaled dose is less than the bloodAUC₀₋₂₄ of up to 801 mg of an orally administered dosage ofitraconazole. In some embodiments, the blood AUC₀₋₂₄ of itraconazolefrom each inhaled dose is less than 80%, less than 70%, less than 60%,less than 50%, less than 40%, less than 30%, less than 20%, less than10%, less than 5%, less than 2.5%, less than 1.0%, less than 0.5%, lessthan 0.25%, less than 0.1%, less than 0.05%, less than 0.025% or lessthan 0.01% of the blood AUC₀₋₂₄ of up to 801 mg of an orallyadministered dosage of itraconazole. In some embodiments, the bloodAUC₀₋₂₄ of itraconazole from each inhaled dose is between 0.01-90%,0.01-80%, 0.01-70%, 0.01-60%, 0.01-50%, 0.01-40%, 0.01-30%, 0.01-20%,0.01-10%, 0.01-5%, 0.01-2.5%, 0.01-1%, 0.01-0.1%, 5-90%, between 5-80%,between 5-70%, between 5-60%, between 5-50%, between 5-40%, between5-30%, between 5-20%, between 5-10%, between 1-5%, between 1-10%,between 1-20%, between 1-30%, between 1-40%, between 1-50%, between1-60%, between 1-70%, between 1-80%, or between 1-90% of the bloodAUC₀₋₂₄ of up to 801 mg of an orally administered dosage ofitraconalzole. In some embodiments, wherein each inhaled dose is lessthan ½ of the up to 801 mg of an orally administered dosage ofitraconazole. In some embodiments, wherein each inhaled dose is lessthan ½, ⅓, ¼, ⅕, ⅙, ⅛, 1/10, 1/20, 1/40, 1/50, 1/75, 1/100, 1/200,1/300, or 1/400 of the up to 801 mg of an orally administered dosage ofitraconazole.

Nanoparticulate Compositions

Another embodiment is directed to dry powders which containnanoparticulate compositions for pulmonary or nasal delivery. Thepowders may consist of respirable aggregates of nanoparticulate drugparticles, or of respirable particles of a diluent which contains atleast one embedded drug nanoparticle. Powders containing nanoparticulatedrug particles can be prepared from aqueous dispersions of nanoparticlesby removing the water via spray-drying or lyophilization (freezedrying). Spray-drying is less time consuming and less expensive thanfreeze-drying, and therefore more cost-effective. However, certaindrugs, such as biologicals benefit from lyophilization rather thanspray-drying in making dry powder formulations.

Conventional micronized drug particles used in dry powder aerosoldelivery having particle diameters of from about 1 to about 5 micronsMMAD are often difficult to meter and disperse in small quantitiesbecause of the electrostatic cohesive forces inherent in such powders.These difficulties can lead to loss of drug substance to the deliverydevice as well as incomplete powder dispersion and sub-optimal deliveryto the lung. Many drug compounds, particularly proteins and peptides,are intended for deep lung delivery and systemic absorption. Since theaverage particle sizes of conventionally prepared dry powders areusually in the range of from about 1 to about 5 microns MMAD, thefraction of material which actually reaches the alveolar region may bequite small. Thus, delivery of micronized dry powders to the lung,especially the alveolar region, is generally very inefficient because ofthe properties of the powders themselves.

The dry powder aerosols which contain nanoparticulate drugs can be madesmaller than comparable micronized drug substance and, therefore, areappropriate for efficient delivery to the deep lung. Moreover,aggregates of nanoparticulate drugs are spherical in geometry and havegood flow properties, thereby aiding in dose metering and deposition ofthe administered composition in the lung or nasal cavities.

Dry nanoparticulate compositions can be used in both DPIs and pMDIs. Asused herein, “dry” refers to a composition having less than about 5%water.

In one embodiment, compositions are provided containing nanoparticleswhich have an effective average particle size of less than about 1000nm, more preferably less than about 400 nm, less than about 300 nm, lessthan about 250 nm, or less than about 200 nm, as measured bylight-scattering methods. By “an effective average particle size of lessthan about 1000 nm” it is meant that at least 50% of the drug particleshave a weight average particle size of less than about 1000 nm whenmeasured by light scattering techniques. Preferably, at least 70% of thedrug particles have an average particle size of less than about 1000 nm,more preferably at least 90% of the drug particles have an averageparticle size of less than about 1000 nm, and even more preferably atleast about 95% of the particles have a weight average particle size ofless than about 1000 nm.

For aqueous aerosol formulations, the nanoparticulate itraconazole maybe present at a concentration of about 34 mg/mL up to about 463 mg/mL.For dry powder aerosol formulations, the nanoparticulate agent may bepresent at a concentration of about 34 mg/g up to about 463 mg/g,depending on the desired drug dosage. Concentrated nanoparticulateaerosols, defined as containing a nanoparticulate drug at aconcentration of about 34 mg/mL up to about 463 mg/mL for aqueousaerosol formulations, and about 34 mg/g up to about 463 mg/g for drypowder aerosol formulations, are specifically provided. Suchformulations provide effective delivery to appropriate areas of the lungor nasal cavities in short administration times, i.e., less than about3-15 seconds per dose as compared to administration times of up to 4 to20 minutes as found in conventional pulmonary nebulizer therapies.

Nanoparticulate drug compositions for aerosol administration can be madeby, for example, (1) nebulizing a dispersion of a nanoparticulate drug,obtained by either grinding or precipitation; (2) aerosolizing a drypowder of aggregates of nanoparticulate drug and surface modifier (theaerosolized composition may additionally contain a diluent); or (3)aerosolizing a suspension of nanoparticulate drug or drug aggregates ina non-aqueous propellant. The aggregates of nanoparticulate drug andsurface modifier, which may additionally contain a diluent, can be madein a non-pressurized or a pressurized non-aqueous system. Concentratedaerosol formulations may also be made via such methods.

Milling of aqueous drug to obtain nanoparticulate drug may be performedby dispersing drug particles in a liquid dispersion medium and applyingmechanical means in the presence of grinding media to reduce theparticle size of the drug to the desired effective average particlesize. The particles can be reduced in size in the presence of one ormore surface modifiers. Alternatively, the particles can be contactedwith one or more surface modifiers after attrition. Other compounds,such as a diluent, can be added to the drug/surface modifier compositionduring the size reduction process. Dispersions can be manufacturedcontinuously or in a batch mode.

Another method of forming nanoparticle dispersion is bymicroprecipitation. This is a method of preparing stable dispersions ofdrugs in the presence of one or more surface modifiers and one or morecolloid stability enhancing surface active agents free of any tracetoxic solvents or solubilized heavy metal impurities. Such a methodcomprises, for example, (1) dissolving the drug in a suitable solventwith mixing; (2) adding the formulation from step (1) with mixing to asolution comprising at least one surface modifier to form a clearsolution; and (3) precipitating the formulation from step (2) withmixing using an appropriate nonsolvent. The method can be followed byremoval of any formed salt, if present, by dialysis or diafiltration andconcentration of the dispersion by conventional means. The resultantnanoparticulate drug dispersion can be utilized in liquid nebulizers orprocessed to form a dry powder for use in a DPI or pMDI.

In a non-aqueous, non-pressurized milling system, a non-aqueous liquidhaving a vapor pressure of about 1 atm or less at room temperature andin which the drug substance is essentially insoluble may be used as awet milling medium to make a nanoparticulate drug composition. In such aprocess, a slurry of drug and surface modifier may be milled in thenon-aqueous medium to generate nanoparticulate drug particles. Examplesof suitable non-aqueous media include ethanol,trichloromonofluoromethane, (CFC-11), and dichlorotetafluoroethane(CFC-114). An advantage of using CFC-11 is that it can be handled atonly marginally cool room temperatures, whereas CFC-114 requires morecontrolled conditions to avoid evaporation. Upon completion of millingthe liquid medium may be removed and recovered under vacuum or heating,resulting in a dry nanoparticulate composition. The dry composition maythen be filled into a suitable container and charged with a finalpropellant. Exemplary final product propellants, which ideally do notcontain chlorinated hydrocarbons, include HFA-134a (tetrafluoroethane)and HFA-227 (heptafluoropropane). While non-chlorinated propellants maybe preferred for environmental reasons, chlorinated propellants may alsobe used in this embodiment of the invention.

In a non-aqueous, pressurized milling system, a non-aqueous liquidmedium having a vapor pressure significantly greater than 1 atm at roomtemperature may be used in the milling process to make nanoparticulatedrug compositions. If the milling medium is a suitable halogenatedhydrocarbon propellant, the resultant dispersion may be filled directlyinto a suitable pMDI container. Alternately, the milling medium can beremoved and recovered under vacuum or heating to yield a drynanoparticulate composition. This composition can then be filled into anappropriate container and charged with a suitable propellant for use ina pMDI.

Spray drying is a process used to obtain a powder containingnanoparticulate drug particles following particle size reduction of thedrug in a liquid medium. In general, spray-drying may be used when theliquid medium has a vapor pressure of less than about 1 atm at roomtemperature. A spray-dryer is a device which allows for liquidevaporation and drug powder collection. A liquid sample, either asolution or suspension, is fed into a spray nozzle. The nozzle generatesdroplets of the sample within a range of about 20 to about 100 micron indiameter which are then transported by a carrier gas into a dryingchamber. The carrier gas temperature is typically from about 80 to about200° C. The droplets are subjected to rapid liquid evaporation, leavingbehind dry particles which are collected in a special reservoir beneatha cyclone apparatus. Smaller particles in the range down about 1 micronto about 5 microns are also possible.

If the liquid sample consists of an aqueous dispersion of nanoparticlesand surface modifier, the collected product will consist of sphericalaggregates of the nanoparticulate drug particles. If the liquid sampleconsists of an aqueous dispersion of nanoparticles in which an inertdiluent material was dissolved (such as lactose or mannitol), thecollected product will consist of diluent (e.g., lactose or mannitol)particles which contain embedded nanoparticulate drug particles. Thefinal size of the collected product can be controlled and depends on theconcentration of nanoparticulate drug and/or diluent in the liquidsample, as well as the droplet size produced by the spray-dryer nozzle.Collected products may be used in conventional DPIs for pulmonary ornasal delivery, dispersed in propellants for use in pMDIs, or theparticles may be reconstituted in water for use in nebulizers.

In some instances it may be desirable to add an inert carrier to thespray-dried material to improve the metering properties of the finalproduct. This may especially be the case when the spray dried powder isvery small (less than about 5 micron) or when the intended dose isextremely small, whereby dose metering becomes difficult. In general,such carrier particles (also known as bulking agents) are too large tobe delivered to the lung and simply impact the mouth and throat and areswallowed. Such carriers typically consist of sugars such as lactose,mannitol, or trehalose. Other inert materials, including polysaccharidesand cellulosics, may also be useful as carriers.

Spray-dried powders containing nanoparticulate drug particles may usedin conventional DPIs, dispersed in propellants for use in pMDIs, orreconstituted in a liquid medium for use with nebulizers.

To avoid denaturization or destabilization by heat, sublimation ispreferred over evaporation to obtain a dry powder nanoparticulate drugcomposition. This is because sublimation avoids the high processtemperatures associated with spray-drying. In addition, sublimation,also known as freeze-drying or lyophilization, can increase the shelfstability of drug compounds, particularly for biological products.Freeze-dried particles can also be reconstituted and used in nebulizers.Aggregates of freeze-dried nanoparticulate drug particles can be blendedwith either dry powder intermediates or used alone in DPIs and pMDIs foreither nasal or pulmonary delivery.

Sublimation involves freezing the product and subjecting the sample tostrong vacuum conditions. This allows for the formed ice to betransformed directly from a solid state to a vapor state. Such a processis highly efficient and, therefore, provides greater yields thanspray-drying. The resultant freeze-dried product contains drug andmodifier(s). The drug is typically present in an aggregated state andcan be used for inhalation alone (either pulmonary or nasal), inconjunction with diluent materials (lactose, mannitol, etc.), in DPIs orpMDIs, or reconstituted for use in a nebulizer.

Liposomal Compositions

In some embodiments, itraconazole may be formulated into liposomeparticles, which can then be aerosolized for inhaled delivery. Lipidswhich are useful in the present invention can be any of a variety oflipids including both neutral lipids and charged lipids. Carrier systemshaving desirable properties can be prepared using appropriatecombinations of lipids, targeting groups and circulation enhancers.Additionally, the compositions provided herein can be in the form ofliposomes or lipid particles, preferably lipid particles. As usedherein, the term “lipid particle” refers to a lipid bilayer carrierwhich “coats” a nucleic acid and has little or no aqueous interior. Moreparticularly, the term is used to describe a self-assembling lipidbilayer carrier in which a portion of the interior layer comprisescationic lipids which form ionic bonds or ion-pairs with negativecharges on the nucleic acid (e.g., a plasmid phosphodiester backbone).The interior layer can also comprise neutral or fusogenic lipids and, insome embodiments, negatively charged lipids. The outer layer of theparticle will typically comprise mixtures of lipids oriented in atail-to-tail fashion (as in liposomes) with the hydrophobic tails of theinterior layer. The polar head groups present on the lipids of the outerlayer will form the external surface of the particle.

Liposomal bioactive agents can be designed to have a sustainedtherapeutic effect or lower toxicity allowing less frequentadministration and an enhanced therapeutic index. Liposomes are composedof bilayers that entrap the desired pharmaceutical. These can beconfigured as multilamellar vesicles of concentric bilayers with thepharmaceutical trapped within either the lipid of the different layersor the aqueous space between the layers.

By non-limiting example, lipids used in the compositions may besynthetic, semi-synthetic or naturally-occurring lipids, includingphospholipids, tocopherols, steroids, fatty acids, glycoproteins such asalbumin, negatively-charged lipids and cationic lipids. Phosholipidsinclude egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG),egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS),phosphatidylethanolamine (EPE), and egg phosphatidic acid (EPA); thesoya counterparts, soy phosphatidylcholine (SPC); SPG, SPS, SPI, SPE,and SPA; the hydrogenated egg and soya counterparts (e.g., HEPC, HSPC),other phospholipids made up of ester linkages of fatty acids in the 2and 3 of glycerol positions containing chains of 12 to 26 carbon atomsand different head groups in the 1 position of glycerol that includecholine, glycerol, inositol, serine, ethanolamine, as well as thecorresponding phosphatidic acids. The chains on these fatty acids can besaturated or unsaturated, and the phospholipid can be made up of fattyacids of different chain lengths and different degrees of unsaturation.In particular, the compositions of the formulations can includedipalmitoylphosphatidylcholine (DPPC), a major constituent ofnaturally-occurring lung surfactant as well asdioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol(DOPG). Other examples include dimyristoylphosphatidycholine (DMPC) anddimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidcholine(DPPC) and dipalmitoylphosphatidylglycerol (DPPG)distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol(DSPG), dioleylphosphatidylethanolamine (DOPE) and mixed phospholipidslike palmitoylstearoylphosphatidylcholine (PSPC) andpalmitoylstearoylphosphatidylglycerol (PSPG), and single acylatedphospholipids like mono-oleoyl-phosphatidylethanolamine (MOPE).

In a preferred embodiment, PEG-modified lipids are incorporated into thecompositions of the present invention as the aggregation-preventingagent. The use of a PEG-modified lipid positions bulky PEG groups on thesurface of the liposome or lipid carrier and prevents binding of DNA tothe outside of the carrier (thereby inhibiting cross-linking andaggregation of the lipid carrier). The use of a PEG-ceramide is oftenpreferred and has the additional advantages of stabilizing membranebilayers and lengthening circulation lifetimes. Additionally,PEG-ceramides can be prepared with different lipid tail lengths tocontrol the lifetime of the PEG-ceramide in the lipid bilayer. In thismanner, “programmable” release can be accomplished which results in thecontrol of lipid carrier fusion. For example, PEG-ceramides havingC20-acyl groups attached to the ceramide moiety will diffuse out of alipid bilayer carrier with a half-life of 22 hours. PEG-ceramides havingC14- and C8-acyl groups will diffuse out of the same carrier withhalf-lives of 10 minutes and less than 1 minute, respectively. As aresult, selection of lipid tail length provides a composition in whichthe bilayer becomes destabilized (and thus fusogenic) at a known rate.Though less preferred, other PEG-lipids or lipid-polyoxyethyleneconjugates are useful in the present compositions. Examples of suitablePEG-modified lipids include PEG-modified phosphatidylethanolamine andphosphatidic acid, PEG-modified diacylglycerols and dialkylglycerols,PEG-modified dialkylamines and PEG-modified1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-ceramideconjugates (e.g., PEG-Cer-C8, PEG-Cer-C14 or PEG-Cer-C20) which aredescribed in U.S. Pat. No. 5,820,873, incorporated herein by reference.

The compositions of the present invention can be prepared to provideliposome compositions which are about 50 nm to about 400 nm in diameter.One with skill in the art will understand that the size of thecompositions can be larger or smaller depending upon the volume which isencapsulated. Thus, for larger volumes, the size distribution willtypically be from about 80 nm to about 300 nm.

Surface Modifiers

Itraconazole may be prepared in a pharmaceutical composition withsuitable surface modifiers which may be selected from known organic andinorganic pharmaceutical excipients. Such excipients include lowmolecular weight oligomers, polymers, surfactants and natural products.Preferred surface modifiers include nonionic and ionic surfactants. Twoor more surface modifiers can be used in combination.

Representative examples of surface modifiers include cetyl pyridiniumchloride, gelatin, casein, lecithin (phosphatides), dextran, glycerol,gum acacia, cholesterol, tragacanth, stearic acid, benzalkoniumchloride, calcium stearate, glycerol monostearate, cetostearyl alcohol,cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkylethers (e.g., macrogol ethers such as cetomacrogol 1000),polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fattyacid esters (e.g., the commercially available Tweens™, such as e.g.,Tween 20™, and Tween 80™, (ICI Specialty Chemicals)); polyethyleneglycols (e.g., Carbowaxs 3350™, and 1450™, and Carbopol 934™, (UnionCarbide)), dodecyl trimethyl ammonium bromide, polyoxyethylenestearates,colloidal silicon dioxide, phosphates, sodium dodecylsulfate,carboxymethylcellulose calcium, hydroxypropyl cellulose (HPC, HPC-SL,and HPC-L), hydroxypropyl methylcellulose (HPMC), carboxymethylcellulosesodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethyl-cellulose phthalate, noncrystalline cellulose,magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA),polyvinylpyrrolidone (PVP), 4-(1,1,3,3-tetaamethylbutyl)-phenol polymerwith ethylene oxide and formaldehyde (also known as tyloxapol,superione, and triton), poloxamers (e.g., Pluronics F68™, and F108™,which are block copolymers of ethylene oxide and propylene oxide);poloxamines (e.g., Tetronic 908™, also known as Poloxamine 908™, whichis a tetrafunctional block copolymer derived from sequential addition ofpropylene oxide and ethylene oxide to ethylenediamine (BASF WyandotteCorporation, Parsippany, N.J.)); a charged phospholipid such asdimyristoyl phophatidyl glycerol, dioctylsulfosuccinate (DOSS); Tetronic1508™; (T-1508) (BASF Wyandotte Corporation), dialkylesters of sodiumsulfosuccinic acid (e.g., Aerosol OT™, which is a dioctyl ester ofsodium sulfosuccinic acid (American Cyanamid)); Duponol P™, which is asodium lauryl sulfate (DuPont); Tritons X-200™, which is an alkyl arylpolyether sulfonate (Rohm and Haas); Crodestas F-110™, which is amixture of sucrose stearate and sucrose distearate (Croda Inc.);p-isononylphenoxypoly-(glycidol), also known as Olin-Log™, or Surfactant10-G™, (Olin Chemicals, Stamford, Conn.); Crodestas SL-40™, (Croda,Inc.); and SA9OHCO, which is C.sub.18H.sub.37CH.sub.2(CON(CH.sub.3)—CH.sub.2(CHOH). sub.4(CH.sub.20H).sub.2 (Eastman KodakCo.); decanoyl-N-methylglucamide; n-decyl .beta.-D-glucopyranoside;n-decyl .beta.-D-maltopyranoside; n-dodecyl .beta.-D-glucopyranoside;n-dodecyl .beta.-D-maltoside; heptanoyl-N-methylglucamide;n-heptyl-.beta.-D-glucopyranoside; n-heptyl .beta.-D-thioglucoside;n-hexyl .beta.-D-glucopyranoside; nonanoyl-N-methylglucamide; n-noyl.beta.-D-glucopyranoside; octanoyl-N-methylglucamide;n-octyl-.beta.-D-glucopyranoside; octyl.beta.-D-thioglucopyranoside; andthe like. Tyloxapol is a particularly preferred surface modifier for thepulmonary or intranasal delivery of steroids, even more so fornebulization therapies.

Examples of surfactants for use in the solutions disclosed hereininclude, but are not limited to, ammonium laureth sulfate, cetamineoxide, cetrimonium chloride, cetyl alcohol, cetyl myristate, cetylpalmitate, cocamide DEA, cocamidopropyl betaine, cocamidopropylamineoxide, cocamide MEA, DEA lauryl sulfate, di-stearyl phthalic acid amide,dicetyl dimethyl ammonium chloride, dipalmitoylethyl hydroxethylmonium,disodium laureth sulfosuccinate, di(hydrogenated) tallow phthalic acid,glyceryl dilaurate, glyceryl distearate, glyceryl oleate, glycerylstearate, isopropyl myristate nf, isopropyl palmitate nf, lauramide DEA,lauramide MEA, lauramide oxide, myristamine oxide, octyl isononanoate,octyl palmitate, octyldodecyl neopentanoate, olealkonium chloride, PEG-2stearate, PEG-32 glyceryl caprylate/caprate, PEG-32 glyceryl stearate,PEG-4 and PEG-150 stearate & distearate, PEG-4 to PEG-150 laurate &dilaurate, PEG-4 to PEG-150 oleate & dioleate, PEG-7 glyceryl cocoate,PEG-8 beeswax, propylene glycol stearate, sodium C14-16 olefinsulfonate, sodium lauryl sulfoacetate, sodium lauryl sulphate, sodiumtrideceth sulfate, stearalkonium chloride, stearamide oxide,TEA-dodecylbenzene sulfonate, TEA lauryl sulfate.

Most of these surface modifiers are known pharmaceutical excipients andare described in detail in the Handbook of Pharmaceutical Excipients,published jointly by the American Pharmaceutical Association and ThePharmaceutical Society of Great Britain (The Pharmaceutical Press,1986), specifically incorporated by reference. The surface modifiers arecommercially available and/or can be prepared by techniques known in theart. The relative amount of drug and surface modifier can vary widelyand the optimal amount of the surface modifier can depend upon, forexample, the particular drug and surface modifier selected, the criticalmicelle concentration of the surface modifier if it forms micelles, thehydrophilic-lipophilic-balance (HLB) of the surface modifier, themelting point of the surface modifier, the water solubility of thesurface modifier and/or drug, the surface tension of water solutions ofthe surface modifier, etc.

In the present invention, the optimal ratio of drug to surface modifieris about 0.1% to about 99.9% itraconazole, more preferably about 10% toabout 90%.

Microspheres

Microspheres can be used for pulmonary delivery of itraconazole by firstadding an appropriate amount of drug compound to be solubilized inwater. For example, an aqueous itraconazole solution may be dispersed inmethylene chloride containing a predetermined amount (0.1-1% w/v) ofpoly(DL-lactide-co-glycolide) (PLGA) by probe sonication for 1-3 min onan ice bath. Separately, an itraconazole may be solubilized in methylenechloride containing PLGA (0.1-1% w/v). The resulting water-in-oilprimary emulsion or the polymer/drug solution will be dispersed in anaqueous continuous phase consisting of 1-2% polyvinyl alcohol(previously cooled to 4° C.) by probe sonication for 3-5 min on an icebath. The resulting emulsion will be stirred continuously for 2-4 hoursat room temperature to evaporate methylene chloride. Microparticles thusformed will be separated from the continuous phase by centrifuging at8000-10000 rpm for 5-10 min. Sedimented particles will be washed thricewith distilled water and freeze dried. Freeze-dried itraconazolemicroparticles will be stored at −20° C.

By non-limiting example, a spray drying approach will be employed toprepare itraconazole microspheres. An appropriate amount of itraconazolewill be solubilized in methylene chloride containing PLGA (0.1-1%). Thissolution will be spray dried to obtain the microspheres.

By non-limiting example, itraconazole microparticles will becharacterized for size distribution (requirement: 90%<5 μm, 95%<10 μm),shape, drug loading efficiency and drug release using appropriatetechniques and methods.

By non-limiting example, this approach may also be used to sequester andimprove the water solubility of solid, AUC shape-enhancing formulations.

A certain amount of itraconazole can be first dissolved in the minimalquantity of ethanol 96% necessary to maintain the fluoroquinolone insolution when diluted with water from 96 to 75%. This solution can thenbe diluted with water to obtain a 75% ethanol solution and then acertain amount of paracetamol can be added to obtain the following w/wdrug/polymer ratios: 1:2, 1:1, 2:1, 3:1, 4:1, 6:1, 9:1, and 19:1. Thesefinal solutions are spray-dried under the following conditions: feedrate, 15 mL/min; inlet temperature, 110° C.; outlet temperature, 85° C.;pressure 4 bar and throughput of drying air, 35 m3/hr. Powder is thencollected and stored under vacuum in a desiccator.

Solid Lipid Particles

Preparation of itraconazole solid lipid particles may involve dissolvingthe drug in a lipid melt (phospholipids such as phophatidyl choline andphosphatidyl serine) maintained at least at the melting temperature ofthe lipid, followed by dispersion of the drug-containing melt in a hotaqueous surfactant solution (typically 1-5% w/v) maintained at least atthe melting temperature of the lipid. The coarse dispersion will behomogenized for 1-10 min using a Microfluidizer® to obtain ananoemulsion. Cooling the nanoemulsion to a temperature between 4-25° C.will re-solidify the lipid, leading to formation of solid lipidnanoparticles. Optimization of formulation parameters (type of lipidmatrix, surfactant concentration and production parameters) will beperformed so as to achieve a prolonged drug delivery. By non-limitingexample, this approach may also be used to sequester and improve thewater solubility of solid, AUC shape-enhancing formulations fornanoparticle-based formulations.

Melt-Extrusion AUC Shape-Enhancing Formulation

Melt-Extrusion AUC shape-enhancing itraconazole formulations may bepreparation by dissolving the drugs in micelles by adding surfactants orpreparing micro-emulsion, forming inclusion complexes with othermolecules such as cyclodextrins, forming nanoparticles of the drugs, orembedding the amorphous drugs in a polymer matrix. Embedding the drughomogeneously in a polymer matrix produces a solid dispersion. Soliddispersions can be prepared in two ways: the solvent method and the hotmelt method. The solvent method uses an organic solvent wherein the drugand appropriate polymer are dissolved and then (spray) dried. The majordrawbacks of this method are the use of organic solvents and the batchmode production process. The hot melt method uses heat in order todisperse or dissolve the drug in an appropriate polymer. Themelt-extrusion process is an optimized version of the hot melt method.The advantage of the melt-extrusion approach is lack of organic solventand continuous production process. As the melt-extrusion is a novelpharmaceutical technique, the literature dealing with it is limited. Thetechnical set-up involves a mixture and extrusion of itraconazole,hydroxypropyl-b-cyclodextrin (HP-b-CD), and hydroxypropylmethylcellulose(HPMC), in order to, by non-limiting example create a AUCshape-enhancing formulation of itraconazole. Cyclodextrin is atoroidal-shaped molecule with hydroxyl groups on the outer surface and acavity in the center. Cyclodextrin sequesters the drug by forming aninclusion complex. The complex formation between cyclodextrins and drugshas been investigated extensively. It is known that water-solublepolymer interacts with cyclodextrin and drug in the course of complexformation to form a stabilized complex of drug and cyclodextrinco-complexed with the polymer. This complex is more stable than theclassic cyclodextrin-drug complex. As one example, HPMC is watersoluble; hence using this polymer with HP-b-CD in the melt is expectedto create an aqueous soluble AUC shape-enhancing formulation. Bynon-limiting example, this approach may also be used to sequester andimprove the water solubility of solid, AUC shape-enhancing formulations,for nanoparticle-based formulations.

Co-Precipitates

Co-precipitate itraconazole formulations may be prepared by formation ofco-precipitates with pharmacologically inert, polymeric materials. Ithas been demonstrated that the formation of molecular solid dispersionsor co-precipitates to create an AUC shape-enhancing formulations withvarious water-soluble polymers can significantly slow the in vitrodissolution rates and/or in vivo absorption. In preparing powderedproducts, grinding is generally used for reducing particle size, sincethe dissolution rate is strongly affected by particle size. Moreover, astrong force (such as grinding) may increase the surface energy andcause distortion of the crystal lattice as well as reducing particlesize. Co-grinding itraconazole with hydroxypropylmethylcellulose,β-cyclodextrin, chitin and chitosan, crystalline cellulose, and gelatin,may enhance the dissolution properties such that AUC shape-enhancementis obtained. By non-limiting example, this approach may also be used tosequester and improve the water solubility of solid, AUC shape-enhancingformulations.

Dispersion-Enhancing Peptides

Compositions may include one or more di- or tripeptides containing twoor more leucine residues. By further non-limiting example, U.S. Pat. No.6,835,372 disclosing dispersion-enhancing peptides, is herebyincorporated by reference in its entirety. This patent describes thediscovery that di-leucyl-containing dipeptides (e.g., dileucine) andtripeptides are superior in their ability to increase the dispersibilityof powdered composition.

In another embodiment, highly dispersible particles including an aminoacid are administered. Hydrophobic amino acids are preferred. Suitableamino acids include naturally occurring and non-naturally occurringhydrophobic amino acids. Some naturally occurring hydrophobic aminoacids, including but not limited to, non-naturally occurring amino acidsinclude, for example, beta-amino acids. Both D, L and racemicconfigurations of hydrophobic amino acids can be employed. Suitablehydrophobic amino acids can also include amino acid analogs. As usedherein, an amino acid analog includes the D or L configuration of anamino acid having the following formula: —NH—CHR—CO—, wherein R is analiphatic group, a substituted aliphatic group, a benzyl group, asubstituted benzyl group, an aromatic group or a substituted aromaticgroup and wherein R does not correspond to the side chain of anaturally-occurring amino acid. As used herein, aliphatic groups includestraight chained, branched or cyclic C1-C8 hydrocarbons which arecompletely saturated, which contain one or two heteroatoms such asnitrogen, oxygen or sulfur and/or which contain one or more units ofdesaturation. Aromatic groups include carbocyclic aromatic groups suchas phenyl and naphthyl and heterocyclic aromatic groups such asimidazolyl, indolyl, thienyl, furanyl, pyridyl, pyranyl, oxazolyl,benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl and acridintyl.

Suitable substituents on an aliphatic, aromatic or benzyl group include—OH, halogen (—Br, —Cl, —I and —F)—O (aliphatic, substituted aliphatic,benzyl, substituted benzyl, aryl or substituted aryl group), —CN, —NO₂,—COOH, —NH₂, —NH (aliphatic group, substituted aliphatic, benzyl,substituted benzyl, aryl or substituted aryl group), —N(aliphatic group,substituted aliphatic, benzyl, substituted benzyl, aryl or substitutedaryl group)₂, —COO(aliphatic group, substituted aliphatic, benzyl,substituted benzyl, aryl or substituted aryl group), —CONH₂,—CONH(aliphatic, substituted aliphatic group, benzyl, substitutedbenzyl, aryl or substituted aryl group)), —SH, —S(aliphatic, substitutedaliphatic, benzyl, substituted benzyl, aromatic or substituted aromaticgroup) and —NH—C(.dbd.NH)—NH₂. A substituted benzylic or aromatic groupcan also have an aliphatic or substituted aliphatic group as asubstituent. A substituted aliphatic group can also have a benzyl,substituted benzyl, aryl or substituted aryl group as a substituent. Asubstituted aliphatic, substituted aromatic or substituted benzyl groupcan have one or more substituents. Modifying an amino acid substituentcan increase, for example, the lypophilicity or hydrophobicity ofnatural amino acids which are hydrophilic.

A number of the suitable amino acids, amino acids analogs and saltsthereof can be obtained commercially. Others can be synthesized bymethods known in the art.

Hydrophobicity is generally defined with respect to the partition of anamino acid between a nonpolar solvent and water. Hydrophobic amino acidsare those acids which show a preference for the nonpolar solvent.Relative hydrophobicity of amino acids can be expressed on ahydrophobicity scale on which glycine has the value 0.5. On such ascale, amino acids which have a preference for water have values below0.5 and those that have a preference for nonpolar solvents have a valueabove 0.5. As used herein, the term hydrophobic amino acid refers to anamino acid that, on the hydrophobicity scale, has a value greater orequal to 0.5, in other words, has a tendency to partition in thenonpolar acid which is at least equal to that of glycine.

Examples of amino acids which can be employed include, but are notlimited to: glycine, proline, alanine, cysteine, methionine, valine,leucine, tyosine, isoleucine, phenylalanine, tryptophan. Preferredhydrophobic amino acids include leucine, isoleucine, alanine, valine,phenylalanine and glycine. Combinations of hydrophobic amino acids canalso be employed. Furthermore, combinations of hydrophobic andhydrophilic (preferentially partitioning in water) amino acids, wherethe overall combination is hydrophobic, can also be employed.

The amino acid can be present in the particles of the invention in anamount of at least 10 weight %. Preferably, the amino acid can bepresent in the particles in an amount ranging from about 20 to about 80weight %. The salt of a hydrophobic amino acid can be present in theparticles of the invention in an amount of at least 10 weight percent.Preferably, the amino acid salt is present in the particles in an amountranging from about 20 to about 80 weight %. In preferred embodiments theparticles have a tap density of less than about 0.4 g/cm³.

Methods of forming and delivering particles which include an amino acidare described in U.S. Pat. No. 6,586,008, entitled Use of Simple AminoAcids to Form Porous Particles During Spray Drying, the teachings ofwhich are incorporated herein by reference in their entirety.

Proteins/Amino Acids

Protein excipients may include albumins such as human serum albumin(HSA), recombinant human albumin (rHA), gelatin, casein, hemoglobin, andthe like. Suitable amino acids (outside of the dileucyl-peptides of theinvention), which may also function in a buffering capacity, includealanine, glycine, arginine, betaine, histidine, glutamic acid, asparticacid, cysteine, lysine, leucine, isoleucine, valine, methionine,phenylalanine, aspartame, tyrosine, tryptophan, and the like. Preferredare amino acids and polypeptides that function as dispersing agents.Amino acids falling into this category include hydrophobic amino acidssuch as leucine, valine, isoleucine, tryptophan, alanine, methionine,phenylalanine, tyrosine, histidine, and proline.Dispersibility-enhancing peptide excipients include dimers, trimers,tetramers, and pentamers comprising one or more hydrophobic amino acidcomponents such as those described above.

Carbohydrates

By non-limiting example, carbohydrate excipients may includemonosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), pyranosyl sorbitol, myoinositol, isomalt, trehaloseand the like.

Polymers

By non-limiting example, compositions may also include polymericexcipients/additives, e.g., polyvinylpyrrolidones, derivatizedcelluloses such as hydroxymethylcellulose, hydroxyethylcellulose, andhydroxypropylmethylcellulose, Ficolls (a polymeric sugar),hydroxyethylstarch, dextrates (by non-limiting example cyclodextrins mayinclude, 2-hydroxypropyl-beta-cyclodextrin,2-hydroxypropyl-gamma-cyclodextrin, randomly methylatedbeta-cyclodextrin, dimethyl-alpha-cyclodextrin,dimethyl-beta-cyclodextrin, maltosyl-alpha-cyclodextrin,glucosyl-1-alpha-cyclodextrin, glucosyl-2-alpha-cyclodextrin,alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, andsulfobutylether-beta-cyclodextrin), polyethylene glycols, and pectin mayalso be used.

Highly dispersible particles administered comprise a bioactive agent anda biocompatible, and preferably biodegradable polymer, copolymer, orblend. The polymers may be tailored to optimize differentcharacteristics of the particle including: i) interactions between theagent to be delivered and the polymer to provide stabilization of theagent and retention of activity upon delivery; ii) rate of polymerdegradation and, thereby, rate of drug release profiles; iii) surfacecharacteristics and targeting capabilities via chemical modification;and iv) particle porosity.

Surface eroding polymers such as polyanhydrides may be used to form theparticles. For example, polyanhydrides such aspoly[(p-carboxyphenoxy)hexane anhydride] (PCPH) may be used.Biodegradable polyanhydrides are described in U.S. Pat. No. 4,857,311.Bulk eroding polymers such as those based on polyesters includingpoly(hydroxy acids) also can be used. For example, polyglycolic acid(PGA), polylactic acid (PLA), or copolymers thereof may be used to formthe particles. The polyester may also have a charged or functionalizablegroup, such as an amino acid. In a preferred embodiment, particles withcontrolled release properties can be formed of poly(D,L-lactic acid)and/or poly(DL-lactic-co-glycolic acid) (“PLGA”) which incorporate asurfactant such as dipalmitoyl phosphatidylcholine (DPPC).

Other polymers include polyamides, polycarbonates, polyalkylenes such aspolyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly(ethylene terephthalate), poly vinyl compounds such aspolyvinyl alcohols, polyvinyl ethers, and polyvinyl esters, polymers ofacrylic and methacrylic acids, celluloses and other polysaccharides, andpeptides or proteins, or copolymers or blends thereof. Polymers may beselected with or modified to have the appropriate stability anddegradation rates in vivo for different controlled drug deliveryapplications.

Highly dispersible particles can be formed from functionalized polyestergraft copolymers, as described in Hrkach et al., (1995) Macromolecules,28: 4736-4739; and Hrkach et al., “Poly(L-Lactic acid-co-amino acid)Graft Copolymers: A Class of Functional Biodegradable Biomaterials” inHydrogels and Biodegradable Polymers for Bioapplications, ACS SymposiumSeries No. 627, Raphael M, Ottenbrite et al., Eds., American ChemicalSociety, Chapter 8, pp. 93-101, 1996.

In a preferred embodiment of the invention, highly dispersible particlesincluding a bioactive agent and a phospholipid are administered.Examples of suitable phospholipids include, among others,phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols,phosphatidylserines, phosphatidylinositols and combinations thereof.Specific examples of phospholipids include but are not limited tophosphatidylcholines dipalmitoyl phosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl phosphatidycholine (DSPC),dipalmitoyl phosphatidyl glycerol (DPPG) or any combination thereof.Other phospholipids are known to those skilled in the art. In apreferred embodiment, the phospholipids are endogenous to the lung.

The phospholipid, can be present in the particles in an amount rangingfrom about 0 to about 90 weight %. More commonly, it can be present inthe particles in an amount ranging from about 10 to about 60 weight %.

In another embodiment of the invention, the phospholipids orcombinations thereof are selected to impart controlled releaseproperties to the highly dispersible particles. The phase transitiontemperature of a specific phospholipid can be below, about or above thephysiological body temperature of a patient. Preferred phase transitiontemperatures range from 30° C. to 50° C. (e.g., within +/−10 degrees ofthe normal body temperature of patient). By selecting phospholipids orcombinations of phospholipids according to their phase transitiontemperature, the particles can be tailored to have controlled releaseproperties. For example, by administering particles which include aphospholipid or combination of phospholipids which have a phasetransition temperature higher than the patient's body temperature, therelease of dopamine precursor, agonist or any combination of precursorsand/or agonists can be slowed down. On the other hand, rapid release canbe obtained by including in the particles phospholipids having lowertransition temperatures.

Taste Masking, Flavor, Other

As also described above, itraconazole formulations disclosed herein andrelated compositions, may further include one or more taste-maskingagents such as flavoring agents, inorganic salts (e.g., sodiumchloride), sweeteners, antioxidants, antistatic agents, surfactants(e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”), sorbitan esters,saccharin (e.g., sodium saccharin or other saccharin forms, which asnoted elsewhere herein may be present in certain embodiments at specificconcentrations or at specific molar ratios relative to itraconazole),bicarbonate, cyclodextrins, lipids (e.g., phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines), fatty acidsand fatty esters, steroids (e.g., cholesterol), and chelating agents(e.g., EDTA, zinc and other such suitable cations). Other pharmaceuticalexcipients and/or additives suitable for use in the compositionsaccording to the invention are listed in “Remington: The Science &Practice of Pharmacy”, 19th ed., Williams & Williams, (1995), and in the“Physician's Desk Reference”, 52nd ed., Medical Economics, Montvale,N.J. (1998).

By way of non-limiting example, taste-masking agents in itraconazoleformulations, may include the use of flavorings, sweeteners, and othervarious coating strategies, for instance, sugars such as sucrose,dextrose, and lactose, carboxylic acids, menthol, amino acids or aminoacid derivatives such as arginine, lysine, and monosodium glutamate,and/or synthetic flavor oils and flavoring aromatics and/or naturaloils, extracts from plants, leaves, flowers, fruits, etc. andcombinations thereof. These may include cinnamon oils, oil ofwintergreen, peppermint oils, clover oil, bay oil, anise oil,eucalyptus, vanilla, citrus oil such as lemon oil, orange oil, grape andgrapefruit oil, fruit essences including apple, peach, pear, strawberry,raspberry, cherry, plum, pineapple, apricot, etc. Additional sweetenersinclude sucrose, dextrose, aspartame, acesulfame-K, sucralose andsaccharin (e.g., sodium saccharin or other saccharin forms, which asnoted elsewhere herein may be present in certain embodiments at specificconcentrations or at specific molar ratios relative to itraconazole),organic acids (by non-limiting example citric acid and aspartic acid).Such flavors may be present at from about 0.05 to about 4 percent byweight, and may be present at lower or higher amounts as a factor of oneor more of potency of the effect on flavor, solubility of the flavorant,effects of the flavorant on solubility or other physicochemical orpharmacokinetic properties of other formulation components, or otherfactors.

Another approach to improve or mask the unpleasant taste of an inhaleddrug may be to decrease the drug's solubility, e.g., drugs must dissolveto interact with taste receptors. Hence, to deliver solid forms of thedrug may avoid the taste response and result in the desired improvedtaste affect.

Moreover, taste-masking may be accomplished by creation of lipopilicvesicles. Additional coating or capping agents include dextrates (bynon-limiting example cyclodextrins may include,2-hydroxypropyl-beta-cyclodextrin, 2-hydroxypropyl-gamma-cyclodextrin,randomly methylated beta-cyclodextrin, dimethyl-alpha-cyclodextrin,dimethyl-beta-cyclodextrin, maltosyl-alpha-cyclodextrin,glucosyl-1-alpha-cyclodextrin, glucosyl-2-alpha-cyclodextrin,alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, andsulfobutylether-beta-cyclodextrin), modified celluloses such as ethylcellulose, methyl cellulose, hydroxypropyl cellulose, hydroxylpropylmethyl cellulose, polyalkylene glycols, polyalkylene oxides, sugars andsugar alcohols, waxes, shellacs, acrylics and mixtures thereof.

An alternative according to certain other preferred embodiments is toinclude taste-modifying agents in the itraconazole formulation. Theseembodiments contemplate including in the formulation a taste-maskingsubstance that is mixed with, coated onto or otherwise combined withitraconazole. Inclusion of one or more such agents in these formulationsmay also serve to improve the taste of additional pharmacologicallyactive compounds that are included in the formulations in addition toitraconazole, e.g., another anti-fibrotic agent such as pirfenidone orpyridone analog compounds, or a mucolytic agent. Non-limiting examplesof such taste-modifying substances include acid phospholipids,lysophospholipid, tocopherol polyethyleneglycol succinate, and embonicacid (pamoate). Many of these agents can be used alone or in combinationwith itraconazole or, in separate embodiments, itraconazole for aerosoladministration.

Mucolytic Agents

Methods to produce formulations that combine agents to reduce sputumviscosity during aerosol treatment with itraconazole include thefollowing. These agents can be prepared in fixed combination or beadministered in succession with aerosol itraconazole therapy.

The most commonly prescribed agent is N-acetylcysteine (NAC), whichdepolymerizes mucus in vitro by breaking disulphide bridges betweenmacromolecules. It is assumed that such reduction of sputum tenacityfacilitates its removal from the respiratory tract. In addition, NAC mayact as an oxygen radical scavenger. NAC can be taken either orally or byinhalation. Differences between these two methods of administration havenot been formally studied. After oral administration, NAC is reduced tocysteine, a precursor of the antioxidant glutathione, in the liver andintestine. The antioxidant properties could be useful in preventingdecline of lung function in cystic fibrosis (CF), chronic obstructivepulmonary disease (COPD) or pulmonary fibrotic diseases (e.g.,idiopathic pulmonary fibrosis). Nebulized NAC is commonly prescribed topatients with CF, in particular in continental Europe, in order toimprove expectoration of sputum by reducing its tenacity. The ultimategoal of this is to slow down the decline of lung function in CF.

L-lysine-N-acetylcysteinate (ACC) or Nacystelyn (NAL) is a novelmucoactive agent possessing mucolytic, antioxidant, andanti-inflammatory properties. Chemically, it is a salt of ACC. This drugappears to present an activity superior to its parent molecule ACCbecause of a synergistic mucolytic activity of L-lysine and ACC.Furthermore, its almost neutral pH (6.2) allows its administration inthe lungs with a very low incidence of bronchospasm, which is not thecase for the acidic ACC (pH 2.2). NAL is difficult to formulate in aninhaled form because the required lung dose is very high (approximately2 mg) and the micronized drug is sticky and cohesive and it is thusproblematic to produce a redispersable formulation. NAL was firstdeveloped as a chlorofluorocarbon (CFC) containing metered-dose inhaler(MDI) because this form was the easiest and the fastest to develop tobegin the preclinical and the first clinical studies. NAL MDI delivered2 mg per puff, from which approximately 10% was able to reach the lungsin healthy volunteers. One major inconvenience of this formulation waspatient compliance because as many as 12 puffs were necessary to obtainthe required dose. Furthermore, the progressive removal of CFC gasesfrom medicinal products combined with the problems of coordination metin a large proportion of the patient population (12) have led to thedevelopment of a new galenical form of NAL. A dry powder inhaler (DPI)formulation was chosen to resolve the problems of compliance with MDIsand to combine it with an optimal, reproducible, and comfortable way toadminister the drug to the widest possible patient population, includingyoung children.

The DPI formulation of NAL involved the use of a nonconventional lactose(usually reserved for direct compression of tablets), namely, aroller-dried (RD) anhydrous beta-lactose. When tested in vitro with amonodose DPI device, this powder formulation produces a fine particlefraction (FPF) of at least 30% of the nominal dose, namely three timeshigher than that with MDIs. This approach may be used in combinationwith itraconazole for either co-administration or fixed combinationtherapy.

In addition to mucolytic activity, excessive neutrophil elastaseactivity within airways of cystic fibrosis (CF) patients results inprogressive lung damage. Disruption of disulfide bonds on elastase byreducing agents may modify its enzymatic activity. Three naturallyoccurring dithiol reducing systems were examined for their effects onelastase activity: 1) Escherichia coli thioredoxin (Trx) system, 2)recombinant human thioredoxin (rhTrx) system, and 3) dihydrolipoic acid(DHLA). The Trx systems consisted of Trx, Trx reductase, and NADPH. Asshown by spectrophotometric assay of elastase activity, the two Trxsystems and DHLA inhibited purified human neutrophil elastase as well asthe elastolytic activity present in the soluble phase (sol) of CFsputum. Removal of any of the three Trx system constituents preventedinhibition. Compared with the monothiols N-acetylcysteine and reducedglutathione, the dithiols displayed greater elastase inhibition. Tostreamline Trx as an investigational tool, a stable reduced form ofrhTrx was synthesized and used as a single component. Reduced rhTrxinhibited purified elastase and CF sputum sol elastase without NADPH orTrx reductase. Because Trx and DHLA have mucolytic effects, weinvestigated changes in elastase activity after mucolytic treatment.Unprocessed CF sputum was directly treated with reduced rhTrx, the Trxsystem, DHLA, or DNase. The Trx system and DHLA did not increaseelastase activity, whereas reduced rhTrx treatment increased solelastase activity by 60%. By contrast, the elastase activity after DNasetreatment increased by 190%. The ability of Trx and DHLA to limitelastase activity combined with their mucolytic effects makes thesecompounds potential therapies for CF.

In addition, bundles of F-actin and DNA present in the sputum of cysticfibrosis (CF) patients but absent from normal airway fluid contribute tothe altered viscoelastic properties of sputum that inhibit clearance ofinfected airway fluid and exacerbate the pathology of CF. One approachto alter these adverse properties is to remove these filamentousaggregates using DNase to enzymatically depolymerize DNA to constituentmonomers and gelsolin to sever F-actin to small fragments. The highdensities of negative surface charge on DNA and F-actin suggest that thebundles of these filaments, which alone exhibit a strong electrostaticrepulsion, may be stabilized by multivalent cations such as histones,antimicrobial peptides, and other positively charged molecules prevalentin airway fluid. Furthermore, as a matter-a-fact, it has been observedthat bundles of DNA or F-actin formed after addition of histone H1 orlysozyme are efficiently dissolved by soluble multivalent anions such aspolymeric aspartate or glutamate. Addition of poly-aspartate orpoly-glutamate also disperses DNA and actin-containing bundles in CFsputum and lowers the elastic moduli of these samples to levelscomparable to those obtained after treatment with DNase I or gelsolin.Addition of poly-aspartic acid also increased DNase activity when addedto samples containing DNA bundles formed with histone H1. When added toCF sputum, poly-aspartic acid significantly reduced the growth ofbacteria, suggesting activation of endogenous antibacterial factors.These findings suggest that soluble multivalent anions have potentialalone or in combination with other mucolytic agents to selectivelydissociate the large bundles of charged biopolymers that form in CFsputum.

Hence, NAC, unfractionated heparin, reduced glutathione, dithiols, Trx,DHLA, other monothiols, DNAse, dornase alfa, hypertonic formulations(e.g., osmolalities greater than about 350 mOsmol/kg), multivalentanions such as polymeric aspartate or glutamate, glycosidases and otherexamples listed above can be combined with itraconazole and othermucolytic agents for aerosol administration to improve anti-fibroticand/or anti-inflammatory activity through better distribution fromreduced sputum viscosity, and improved clinical outcome through improvedpulmonary function (from improved sputum mobility and mucociliaryclearance) and decreased lung tissue damage from the immune inflammatoryresponse.

In some embodiments, the method further comprises administration of oneor more additional therapeutic agents to the mammal.

EXAMPLES

Because the relevancy to human efficacy in bleomycin-induced lungfibrosis mouse model, though well established in preclinical studies(Moeller, 2008), has not been established, a new version of thebleomycin-induced lung fibrosis mouse study protocol was designed, withmarketed anti-fibrotic drugs, namely pirfenidone and nintedanib, aspositive controls. Another anti-fungal agent, Fluconazole, was alsotested to show that anti-fungal activities alone cannot reduce lungfibrosis.

Our study showed that treatment of itraconazole alone at 7.5 and 15mg/kg, which led to plasma exposure levels at 394±66 and 899±192 ng/mL,resulted in significant reduction in fibrotic activity, includingreduction in 1) modified Ashcroft score, 2) stained area for collagen,inflammation and αSMA.

Typical dose levels of itraconazole in the clinic are 100 mg (PO/QD),200 mg (PO/QD) or 200 mg (PO/BID). Peak plasma levels are reached 2-5hours post oral dose. As a consequence of non-linear pharmacokinetics,itraconazole accumulates in plasma during multiple dosing. Steady stateplasma levels are generally reached around 15 days post oraladministration. Cmax at steady state were reported at 500 ng/mL at 100mg (PO/QD), 1100 ng/mL at 200 mg (PO/QD) and 2000ng/mL at 200 mg(PO/BID), respectively (Sporanox-itraconazole capsule product label),indicating that efficacious dose levels observed at 7.5 mg/kg and 15mg/kg in our model study are well within the clinically acceptabledoses.

Furthermore, in the same study, it was found that the anti-fibroticactivity of itraconazole is better than that of pirfenidone at 200mg/kg, and comparable if not better than that of nintedanib at 60 mg/kg,an agent recently approved for treating patients with IPF.

Also, the studies have demonstrated that Fluconazole, anotheranti-fungal agent with structural similarity to itraconazole, did notshow detectable changes in fibrosis related endpoint measurements at 70mg/kg daily dose which resulted in plasma level of 56090 ±4947 ng/mL,over 62-fold higher than that of itraconazole at 15 mg/kg.

While an exact mechanism for its anti-fibrotic activity is not clear atthe moment, it is believed that treatment of itraconazole will bebeneficial to patients of IPF, and propose to treat IPF patients withitraconazole either alone or in combination of other agents that maypossess beneficial effects to IPF patients. Itraconazole may beadministered intravenously, intranasal/intratracheally or orally.

Oral dosing of itraconazole were compared to those from variousinhalation formulations of itraconazole in the bleomycin-induced mousemodel, and improved efficacy of itraconazole treatment in mouse modelcan be achieved, which greatly lower the efficacious dose forlung-fibrosis treatment to reduce possible side effects, especially inlong-term uses.

Example 1 Establishment of Positive and Negative Controls

Forty male C57BL/6 mice (Nanjing Biomedical Research Institute ofNanjing University) were randomly divided into two groups on Day 1, 10animals for one group (referred to as Control group or Group 1) and 30for the other. The animals in the Control group were administeredintratracheally with PBS at a dose of 2 mL/kg while the others wereadministered intratracheally with bleomycin (Cat#HY-17565, MCE) at adose of 0.66 mg/kg.

On Day 5, the mice with bleomycin treatment were divided into threegroups at random (referred to as Group 2-4, n=10), and orallyadministered with vehicle (0.5% Methyl cellulose), nintedanib(Kangmanlin Co. Ltd., prepared in 0.5% Methyl cellulose with a finalconcentration of 6.0 mg/mL) and fluconazole (Kangmanlin Co. Ltd.,prepared in 0.5% Methyl cellulose with a final concentration of 7.0mg/mL) at daily doses of 10 mL/kg, 60 mg/kg and 70 mg/kg, respectively.The mice in the Control group was administered with vehicle (0.5% Methylcellulose) at a daily dose of 10 mL/kg. Mice body weights were measuredand recorded on Day 1, 5, 12, 19 and 25.

Two hours post drug administration on Day 25, all animals wereanaesthetized with pentobarbital (i.p., 65 mg/kg). Blood samples werecollected from orbital vein, put in tubes pre-coated with EDTA-K₂ (KANGJIAN, Cat#KJ202), centrifuged at 4000 ×g for 10 minutes at 4° C. andthen stored at −80° C. for determination of plasma drug levels. Then,the trachea was exposed, and bronchoalveolar lavage fluid (BALF) wascollected from each mouse by injecting 0.3 mL of saline into tracheaclose to the larynx for three times. After BALF collection, mice wereperfused with saline, and all four lung lobes were collected from eachmouse, fixed in 10% NBF solution, embedded in paraffin and cut into 5μm-thick sections. The sections were de-paraffinized with xylene andethanol and subject to Haematoxylin-Eosin, Masson Trichrome and α-SMAIHC staining.

The bronchoalveolar lavage fluid of 20 μL was mixed with 20 μL of trypanblue to determine the cell number. Then, the cells in the BALF wereconcentrated using a Cytospinat at 1,000 rpm for 5 minutes, smeared onslides and then stained with Diff-Quik stain. Cell types (alveolarmacrophages, neutrophils, or lymphocytes) were determined by counting atleast 200 cells using a standard hemocytometer. The rest BALFs werecentrifuged and supernatants were collected and stored at −80° C. forsoluble collagen analysis.

The Haematoxylin-Eosin staining was used to identify and quantitate theinflammatory cells in the tissues, and the total inflammation area wascalculated using the AperioScanScope software.

The sections with Masson Trichrome staining were scored for pulmonaryfibrosis according to the modified Ashcroft fibrosis rating criteria inTable 1 below. Each lung lobe was divided into 4 parts according to thedistance from the main bronchus, i.e., the upper, upper middle, lowermiddle and lower parts, which were scored separately and averaged later.

TABLE 1 Modified Ashcroft Score Grade of Fibrosis Characterization ofthe Modified Ashcroft Score 0 Alveolar septa: No fibrotic burden at themost f8msy small fibers in some alveolar walls Lung structure: Normallung 1 Alveolar septa: Isolated gentle fibrotic changes (septum ≤3xthicker than normal) Lung structure: Alveoli partly enlarged andrarefied, but no fibrotic masses present 2 Alveolar septa: Clearlyfibrotic changes (septum >3x thicker than normal) with knot-likeformation but not connected to each other Lung structure: Alveoli partlyenlarged and rarefied, but no fibrotic masses 3 Alveolar septa:Contiguous fibrotic walls (septum >3x thicker than normal) predominantlyin whole microscopic field Lung structure: Alveoli partly enlarged andrarefied, but no fibrotic masses 4 Alveolar septa: Variable Lungstructure: Single fibrotic masses(≤10% of microscopic field) 5 Alveolarsepta: Variable Lung structure: Confluent fibrotic masses(>10% and ≤50%of microscopic field). Lung structure severely damaged but stillpreserved 6 Alveolar septa: Variable, mostly not existent Lungstructure: Large contiguous fibrotic masses(>50% of microscopic field).Lung architecture mostly not preserved 7 Alveolar septa: non-existentLung structure: Alveoli nearly obliterated with fibrous masses but stillup to five air bubbles 8 Alveolar septa: non-existent Lung structure:Microscopic field with complete obliteration with fibrotic masses

α-SMA is a well characterized marker in fibrosis. Immunohistochemistrystaining of α-SMA is often used to characterize and quantitate thetissue fibrosis. Increased staining of 60 -SMA often reflectsaccumulation of collagen-producing myofibroblasts. Before α-SMA IHCstaining, the de-paraffinized sections were treated with citrate antigenretrieval solution (0.01 N citrate buffer, pH 6.0) at 100° C. for 10minutes, and then incubated with rabbit polyclonal anti-α-SMA (1:400,ab5694, Abcam) for 1 hour followed by goat anti-rabbit HRP (K4003,Dako). DAB substrate kit (DAB0031, Maixin, Shanghai) was used forchromogenic staining. All sections were further counter-stained withhematoxylin. Red α-SMA staining areas were calculated using theAperioImageScope program.

Statistical analysis was performed using student's T-test, one-way ortwo-way ANOVA followed by post-hoc Dunnett's test if significant.Non-parametric test like Mann-Whitney was used when N was too small ordata did not follow Gaussian distribution. The difference was consideredsignificant when p<0.05. *p<0.05, ** p<0.01, ***p<0.01.

Mice body weight changes were shown in FIG. 1. Mice in Group 1 had theirbody weights increased as the experiment proceeded while the bodyweights of mice in other groups decreased slightly or remain unchanged.The two-hour post dosing plasma levels were 1079 ng/mL and 56090 ng/mLfor nintedanib and fluconazole, respectively.

Bleomycin caused increase in BALF collagen levels, indicating elevatedinflammatory and fibrotic responses, as shown in FIG. 2, but treatmentwith nintedanib significantly reduced collagen levels. No collagen levelreduction was observed in animals administered with fluconazole.Bleomycin treatment also increased total cell number andmacrophage/monocyte number, and treatment with nintedanib reduced cellnumber to some extent, see FIG. 3.

FIG. 4 (panel A-C) showed the Haematoxylin-Eosin, Masson Trichrome andα-SMA IHC staining analysis results. As expected, administration ofbleomycin resulted in more inflammation areas, higher modified Ashcroftscore and increased α-SMA staining in lungs. Nintedanib administrationsignificantly reduced lung inflammation area, modified Ashcroft scoreand α-SMA staining area (by 84%, 39% and 39%, respectively), which wasnot observed following fluconazole treatment.

It is of critical importance that the results of our anti-IPF efficacystudies are reproduceable and can be compared to those from differentstudies of different drugs in the same animal disease model. By usingnintedanib (a marketed IPF drug) as the positive control and fluconazole(a marketed azole class antifungal drug) as the negative control inBleomycin induced IPF mouse model, the validity of the animal model wasconfirmed, and the study above using the animal model recapitulated theknown anti-IPF efficacies of nintedanib.

In addition, although measured plasma levels passed the antifungalefficacious levels, fluconazole failed to show any significant anti-IPFactivities, whereas nintedanib was efficacious. The results from thisstudy clearly demonstrated that antifungal activities known for thisclass of drugs cannot be translated directly to anti-IPF activities.

Example 2 Oral and Combination Treatment of Itraconazole InhibitedFibrosis

Fifty male C57BL/6 mice (Gempharmatech Co., Ltd.) were randomly dividedinto two groups on Day 1, 10 animals for one group (referred to asControl group or Group 1) and 40 for the other. The animals in theControl group were administered intratracheally with PBS at a dose of 2mL/kg while the others were administered intratracheally with bleomycin(Cat#HY-17565, MCE) at a dose of 0.66 mg/kg.

On Day 5, the mice with bleomycin treatment were divided into fourgroups at random (referred to as Group 2-5, n=10), and orallyadministered with vehicle (DMSO: PEG400=1:9, V/V), itraconazole(Kangmanlin Co. Ltd. prepared in DMSO/PEG400 with a final concentrationof 1.5 mg/mL), nintedanib (Kangmanlin Co. Ltd., prepared in DMSO/PEG400with a final concentration of 6.0 mg/mL), and itraconazole +nintedanib(prepared in DMSO/PEG400 with final concentrations of 1.5 mg/mL and 6.0mg/mL) at daily doses of 10 mL/kg, 15 mg/kg, 60 mg/kg and 15+60 mg/kg,respectively. The mice in the Control group was administered withvehicle (DMSO/PEG400) at a daily dose of 10 mL/kg. Mice body weightswere measured and recorded on Day 1, 5, 12, 19 and 21.

Two hours post drug administration on Day 21, all animals wereanaesthetized with pentobarbital (i.p., 60 mg/kg). Blood sample,bronchoalveolar lavage fluid (BALF) and lung lobes were collected fromeach mouse and then processed following the protocols in Example 1.

The two-hour post dosing plasma levels were 700 ng/mL and 310 ng/mL foritraconazole and nintedanib, respectively. When itraconazole andnintedanib were combined in administration, the plasma drug levels were518 ng/mL and 397 ng/mL, respectively.

Mice body weights during the experiment were recorded and shown in FIG.5. It can be seen that itraconazole and/or nintedanib administrationinhibited bleomycin-induced body weight reduction to some extent.

BALF collagen and cytological analysis results were shown in FIG. 6-7.Compared to the model group, i.e., Group 2, the collagen level andneutrophil number were reduced in animals following itraconazole,nintedanib and combination administration. In addition, itraconazole,nintedanib and combination administration significantly reduced lunginflammation area, modified Ashcroft score and α-SMA staining area ascompared to the model group caused by bleomycin treatment, as shown inFIG. 8 (panel A-C).

Studies on itraconazole and its combination with nintedanib in bleomycininduced IPF mouse model clearly demonstrated that itraconazole washighly efficacious against IPF at its clinically acceptable plasmalevels, and was comparable favorably to marketed anti-IPF drugnintenanib. All indicators, from collagen content in BALF toinflammatory cell counts, to histopathological images, showedsignificant improvement by the end of the study. Based on the negativeresults of fluconazole from the study in Example 1, the activitiesobserved for itraconazole was unlikely from its known antifungalactivities, and a new mode of action must be involved. Further, signs ofimprovement were observed in the combination therapy as compared torespective mono-therapies (see FIG. 6-7), and further studies wereneeded.

Example 3 Inhalation Treatment of Itraconazole Inhibited Fibrosis

Sixty male ICR mice (SHANGHAI SLAC LABORATORY ANIMAL CO., LTD) wererandomly divided into two groups on Day 1, 10 animals for one group(referred to as Control group or Group 1) and 50 for the other. The micewere anesthetized by intraperitoneal injection of 1.5% pentobarbitalsodium solution at a dose of 0.1 mL/20 g, and then supinely positionedand immobilized. Iodine was used to disinfect the neck hair and skin.Later, the neck skin was cut to expose the trachea. 50 μL of PBS orbleomycin hydrochloride (Hisun Pfizer pharmaceutical Co., LTD, 17001711)in 0.9% sodium chloride solution (0.35 USP/ml, i.e., 350 bleomycinunits/mL) was quickly sprayed as mist into the trachea of mice from theControl Group or the other group with a high pressure syringe connectedto a spraying nozzle. At the end, the skin was sutured and disinfected,and the mice were returned to the cages for recovery.

On Day 5, the mice treated with bleomycin were divided into six groupsat random (referred to as Group 2-7). Drug administration was performedfrom Day 5-27 according to the dosing scheme in Table 2 below.

For itraconazole inhalation, each mouse was placed in a 16×13×9(length×width×height in cm) cabinet. Itraconazole preparation of 5, 15or 50 mg/mL was sprayed into the cabinet with a compressed nebulizer.

After 50 mg/mL itraconazole preparation was fully filled in the cabinet,a 50 mL syringe (containing 2 mL of mobile phase for liquidchromatography) was used to suck 48 mL of atomized liquid fordetermination of itraconazole's concentration. The syringe was sealed,stayed still for 10 minutes, sharply shaken and then sent for liquidchromatography (LC). The mobile phase for LC was Acetonitrile-phosphatebuffer (65:35) (6.8 g KH₂PO₄ dissolved in 1000 mL pure water, then pHvalue adjusted to 7.0 with NaOH), and the flow rate was 1.0 mL/min. TheKromasil C18 (4.6×150 mm, 5 μm) was used as the chromatographic columnwith a column temperature of 30° C. Detection wavelength was set at 256nm, and the sample volume for injection was 50 μL.

TABLE 2 Animal grouping and dosing scheme Group Animal no. number DrugDose Treatment 1 10 ethanol 3 min/day inhale, q.d. 2 8 ethanol 3 min/dayinhale, q.d. 3 8 itraconazole hydrochloride (FrontHealth) in 3 min/dayinhale, q.d. ethanol, 5 mg/mL 4 8 itraconazole hydrochloride in ethanol,15 mg/mL 3 min/day inhale, q.d. 5 9 itraconazole hydrochloride inethanol, 50 mg/mL 3 min/day inhale, q.d. 6 8 itraconazole hydrochloridein 0.5% sodium 15 mg/kg body p.o., q.d. carboxymethyl cellulose, 1.5mg/mL weight 7 8 nintedanib (BIBF, Kangmanlin Co., Ltd) in 60 mg/kg bodyp.o., q.d. 0.9% sodium chloride, 6 mg/mL weight

Twenty-four hours after the last drug administration, the mice weresacrificed by dislocation of cervical vertebra. The trachea was isolatedand intubated. The upper lobe of the left lung was ligated. Alveolarlavage was performed with 0.5 mL of alveolar lavage solution for 3times, which was later mixed together and referred to as BALF. The totalnumber of leukocytes in the BALF was counted. The BALF was centrifugedat 2000 rpm/min for 10 min at 4° C. The supernatant was collected andstored at −80° C. for cytokine measurement, and the deposit was smearedon glass slides. After air drying at room temperature, the slides weresubjected to Wright's-Giemsa staining. The number of neutrophils,lymphocytes and macrophages were counted under a microscope from a totalof 200 cells for each slide. The upper left lung tissues were used formeasuring the level of hydroxyproline, and the lower part was fixed informalin for H&E staining and Masson's staining.

The level of hydroxyproline in the lung tissues was measured using aHydroxyproline Assay kit (Nanjing JianCheng Bioengineering Institute).The level of TGF-β1 in 100 μL BALF was determined using a mouse TGF-β1ELISA kit (Beijing 4A Biotech Co., LTD), and the level of type Icollagen (Col I) in 100 μL BALF was determined using a mouse CollagenType I ELISA kit (CUSABIO BIOTEH).

The lower part lung tissues were washed overnight in running water, thengradually dehydrated in 70%-100% ethanol, immersed in xylene andembedded in paraffin. Then, the lung tissues were sectioned into 4μm-thick sections for H&E staining and Masson's staining. H&E stainingwas used to observe the alveolar hyperemia, the infiltration oraggregation of neutrophils in the alveolar space or the vascular wall,and the thickening of alveolar septum or the formation of hyalinemembranes. The degree of fibrosis in the lesions with H&E staining wasobserved under microscope and scored using a semi-quantitative Ashcroftscoring criteria below. Grade 0: Normal tracheal bronchoalveolarstructures; Grade 1: Minimal fibrous thickening of alveolar orbronchiolar walls; Grade 3: Moderate thickening of walls without obviousdamage to lung architecture; Grade 5: Increased fibrosis with definitedamage to lung structure and formation of fibrous bands or small fibrousmasses; Grade 7: Severe distortion of structure and large fibrous areas,with honeycomb lung placed in this category; Grade 8: Total fibrousobliteration. The severity of fibrosis between two odd numbers wasconsidered as the corresponding even number. On the other hand, IODvalue of positive Masson's staining site was measured by Image prosoftware for analysis.

The data were tested for the homogeneity of variance. If the variancewas homogenous (p>0.05), a single factor variance analysis wasperformed. The Dunnet test was performed for the differences betweeneach dose group and the control group. If the variance was heterogenous(p≤0.05), a non-parametric test was conducted, followed by themann-whitney U test for examining the differences between each dosegroup and the control group. The difference was considered significantwhen p<0.05.

Determination of itraconazole concentration by high performance liquidchromatography obtained a standard curve Y=93079X-9553.5, r²≥0.999,where X represented itraconazole concentration and Y represented peakarea, with a faithful linear relationship within the range of 0.1 to114.5 μg/mL. The mean concentration of itraconazole in the mobile phasewas determined to be 69.08 μg/mL, so the itraconazole concentration inthe cabinet was 2.88 μg/mL. Given that mouse's minute ventilation volumewas about 24 mL/min, the daily doses for itraconazole inhalation at 50,15 and 5 mg/mL were calculated to be about 8.3, 2.5 and 0.8 mg/kg,respectively.

The number of total leukocytes, neutrophils, lymphocytes and macrophagesin the BALF increased significantly (p<0.001) on Day 28 due to bleomycintreatment. Itraconazole inhalation treatment at 5, 15 and 50 mg/mL for 3min/day for 22 days reduced the total number of leukocytes in the BALFin a dose dependent manner. Compared with the model group (Group 2),itraconazole inhalation at 50 mg/ml significantly inhibited the increaseof the total number of leukocytes (p<0.05). Itraconazole inhalation at5, 15, 50 mg/mL for 3 min significantly inhibited the increases inneutrophils in the BALF (p<0.05˜0.01), but had no effects on the numberof lymphocytes and macrophages. Oral administration of nintedanib at 60mg/kg as a positive control significantly reduced the total number ofleukocytes and neutrophils in the BALF (p<0.01), but had no effects onthe number of lymphocytes and macrophages. The results were shown inFIG. 9 (panel A and B).

Due to bleomycin treatment, the level of TGF-β1 significantly increasedin the BALF on Day 28 (p<0.001). Itraconazole inhalation at 5, 15, 50mg/mL for 3 min/day for 22 days dose-dependently decreased the level ofTGF-β1 in the BALF. Compared with the model group, the itraconazoleinhalation at 5 mg/mL and 50 mg/mL significantly reduced the level ofTGF-β1 content in the BALF (p<0.05˜0.001). The level of TGF-β1 in theBALF following itraconazole inhalation at 15 mg/mL had a trend ofdecrease but with a large standard deviation. The level of TGF-β1 in theBALF was also significantly reduced by oral administration of nintedanibat 60 mg/kg (p<0.001, FIG. 10, panel A).

With bleomycin treatment, the level of Col I in the BALF increasedsignificantly on Day 28 (p<0.001). Itraconazole inhalation at 5, 15, 50mg/mL for 3 min for 22 days dose-dependently reduced the level of Col Iin the BALF. Compared with the model group, itraconazole inhalation at50 mg/mL significantly reduced the level of Col I in the BALF (p<0.05).Oral administration of nintedanib 60 mg/kg did not reduce the level ofCol I in the BALF (FIG. 10, panel B).

The level of hydroxyproline in the lung tissues increased significantlyon Day 28 (p<0.001). Itraconazole inhalation at 5, 15 and 50 mg/mL for 3min/day for 22 days dose-dependently suppressed the level ofhydroxyproline in the lung tissues. Compared with the model group, thelevel of hydroxyproline in the lung tissues were decreased significantlyin the 15 and 50 mg/mL administration groups (p<0.05˜0.01). Oraladministration of nintedanib at 60 mg/kg significantly reduced the levelof hydroxyproline in the lung tissues (p<0.01). The results were shownin FIG. 11.

On Day 28, lung tissues were performed for H&E staining followed by amicroscopic observation, where an obvious alveolar hyperemia wasobserved. A large number of neutrophils and macrophages infiltratedaround the small airway. Alveolar septum were thickened, hyalinemembrane was formed, and lung tissues were compacted. Itraconazoleinhalation at 15, 50 mg/mL significantly alleviated the pathologicalchanges observed in the mouse model of pulmonary fibrosis, but theinhalation dose of 5 mg/mL did not alleviate the pathological changes.Oral administration of nintedanib at 60 mg/kg significantly reduced theinfiltration of neutrophils and macrophages and the thickening ofalveolar septum in the lung tissues. Semi-quantitative Ashcroft Scoreshowed that itraconazole inhalation at 5, 15, 50 mg/mL dose-dependentlyreduced the pulmonary fibrosis in mice, and oral administration ofnintedanib at 60 mg/kg significantly reduced the pulmonary fibrosis too(p<0.01, FIG. 12).

On Day 28, Masson's staining was done followed by a microscopicobservation and semi-quantitative analysis using image pro software.Collagen deposition in the lungs was observed in the model group whileitraconazole inhalation at 5, 15, 50 mg/mL dose-dependently reduced thecollagen deposition. Compared with the model group, itraconazoleinhalation at 15 or 50 mg/mL significantly reduced the collagendeposition in the lung tissues (p<0.01˜0.001), and oral administrationof nintedanib at 60 mg/kg also significantly reduced the collagendeposition (p<0.001, FIG. 13).

In a separate experiment, three groups of healthy male mice were treatedwith itraconazole for 5 consecutive days. Animals in Group A were orallyadministered with itraconazole in DMSO:PEG400 (1:9, v/v) at a daily doseof 15 mg/kg, while mice in Group B and C were subject to 3-mininhalation treatment of itraconazole of 15 mg/mL and 50 mg/mL,respectively. Two hours after the last dose, plasma samples and lungtissues were collected as described above. These samples were sent to anindependent bioanalytical service lab to determine the levels ofitraconazole, and results were summarized in Table 3.

TABLE 3 Average drug concentrations in plasma and lung tissues GroupPlasma Lung Tissue no. Drug Administration Concentrations ConcentrationsA p.o., 15 mg/kg, q.d. 133 ng/mL 172 ng/mL B i.h., 15 mg/mL for 3 min,q.d. 80.9 ng/mL  590 ng/mL C i.h., 50 mg/mL for 3 min, q.d. 168 ng/mL1396 ng/mL 

After 5 days of oral dosing at 15 mg/kg, itraconazole levels in lungtissues were about 1.3× of that in plasma, showing a moderateaccumulation in lung as compared to systemic circulation.

After 5 days of inhalation at 15 mg/mL for 3 min, itraconazole levels inlung tissues were about 7.3× of that in plasma, showing a significantincrease in lung as result of the direct dosing route. Similarly, at theinhalation dose of 50 mg/mL for 3 min, the differences were 8.3× infavor of lung tissue.

This study was designed to demonstrate that by administeringitraconazole directly to lung tissues via inhalation the safety windowof anti-IPF treatment can be further widened.

The results from this study established that itraconazole administeredvia inhalation is efficacious against bleomycin induced IPF in mice in adose-proportional fashion. All indicators, from collagen content in BALFto inflammatory cell counts, to histopathological images, showedsignificant improvement by the end of the study. Signs of furtherimprovements compared to oral dosing of the same drug were observed.Analysis of lung tissues and plasma samples showed the observedefficacies are more likely driven by the exposures of the drug in lungtissues, but not the exposures of the drug in plasma. After oral dosing,itraconazole levels in lung tissues was slightly higher than those inplasma, making itraconazole the antifungal of choice for lunginfections. However, inhalation greatly enhanced lung tissue exposure,and likely accounted for extra anti-IPF activities observed. Theinhalation formulation and device have not been optimized, and furtherresearch and development is warranted.

Because of the greatly enhanced ratios of lung tissue concentration vs.plasma concentration after inhalation of itraconazole, and its anti-IPFactivities is most likely driven by its exposure in lung tissue, thesafety window of itraconazole through inhalation is likely to beexpanded significantly as compared to oral dosing, and making inhalationa very attractive alternative, especially for chronic treatment, whichis very likely required for most IPF patients.

We claim:
 1. A method for treating idiopathic pulmonary fibrosis,comprising administering to a patient in need thereof, a pharmaceuticalcomposition comprising an effective amount of itraconazole, and apharmaceutically acceptable excipient.
 2. The method of claim 1, whereina daily dose of itraconazole in the range of 20 mg to 1200 mg isadministered to the patient.
 3. The method of claim 1, wherein a dailydose of itraconazole of 1 mg/kg to 20 mg/kg bodyweight is administeredto the patient.
 4. The method of claim 1, wherein itraconazole isadministered by means of an oral, parenteral, rectal, cutaneous, nasal,vaginal, or inhalant route.
 5. The method of claim 1, whereinitraconazole is administered in combination with an effective amount ofanother antifibrosis agent.
 6. The method of claim 5, wherein theantifibrosis agent is pirfenidone or nintedanib.
 7. A pharmaceuticalcomposition for treating IPF comprising an effective amount ofitraconazole, and a pharmaceutically acceptable excipient comprises. 8.The pharmaceutical composition of claim 7, comprising an amount ofitraconazole in the range of 20 mg to 1200 mg.
 9. The pharmaceuticalcomposition of claim 7, further comprising a pharmaceutically effectiveamount of another antifibrosis agent.
 10. The pharmaceutical compositionof claim 9, wherein the antifibrosis agent is pirfenidone or nintedanib.11. A dosage form for delivering itraconazole for treating IPF in apatient in need thereof, wherein the dosage form directly delivers aneffective amount of itraconazole into the lungs of the patient.
 12. Thedosage form of claim 11, wherein it delivers less than about 1/10 of anoral dosage for to the patient.
 13. The dosage form of claim 11, whereinthe dosage form is a spray or a nebulizer.
 14. The dosage form of claim13, further comprising a pharmaceutically effective amount of anantifibrosis agent.
 15. The pharmaceutical composition of claim 14,wherein the antifibrosis agent is pirfenidone or nintedabib.